Microrna compounds and methods for modulating mir-21 activity

ABSTRACT

Described herein are compositions and methods for the inhibition of miR-21 activity. The compositions have certain nucleoside modifications that yield potent inhibitors of miR-21 activity. The compounds may comprise conjugates to facilitate delivery to the liver. The compositions may be administered to subjects with liver conditions, such as liver cancer.

FIELD OF INVENTION

Provided herein are methods and compositions for the modulation ofmiR-21 activity.

DESCRIPTION OF RELATED ART

MicroRNAs (microRNAs), also known as “mature microRNA” are small(approximately 18-24 nucleotides in length), non-coding RNA moleculesencoded in the genomes of plants and animals. In certain instances,highly conserved, endogenously expressed microRNAs regulate theexpression of genes by binding to the 3′-untranslated regions (3′-UTR)of specific mRNAs. More than 1000 different microRNAs have beenidentified in plants and animals. Certain mature microRNAs appear tooriginate from long endogenous primary microRNA transcripts (also knownas pri-microRNAs, pri-mirs, pri-miRs or pri-pre-microRNAs) that areoften hundreds of nucleotides in length (Lee, et al., EMBO J., 2002,21(17), 4663-4670).

Functional analyses of microRNAs have revealed that these smallnon-coding RNAs contribute to different physiological processes inanimals, including developmental timing, organogenesis, differentiation,patterning, embryogenesis, growth control and programmed cell death.Examples of particular processes in which microRNAs participate includestem cell differentiation, neurogenesis, angiogenesis, hematopoiesis,and exocytosis (reviewed by Alvarez-Garcia and Miska, Development, 2005,132, 4653-4662).

SUMMARY OF INVENTION

Provided herein are compounds comprising a modified oligonucleotide,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 and wherein the modified oligonucleotide has anucleoside pattern described herein.

Provided herein are methods for inhibiting the activity of miR-21comprising contacting a cell with a compound described herein. Incertain embodiments, the cell is in vivo. In certain embodiments, thecell is in vitro.

Provided herein are methods for treating a disease associated withmiR-21 comprising administering to a subject having a disease associatedwith miR-21 a compound described herein. In certain embodiments, thesubject is a human. In certain embodiments, the subject is a canine.

The compounds described herein are provided for use in therapy.

Provided herein are compounds comprising modified oligonucleotidescovalently attached to a conjugate moiety. In certain embodiments, acompound has the structure L_(n)-linker-X-MO, wherein each L is,independently, a ligand and n is from 1 to 10; X is a phosphodiesterlinkage or a phosphorothioate linkage; and MO is a modifiedoligonucleotide, wherein the modified oligonucleotide consists of 8 to22 linked nucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to miR-21 (SEQ ID NO: 1).

In certain embodiments, a compound has the structureL_(n)-linker-X₁—N_(m)—X₂-MO, wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; X₁ and X₂ are each,independently, a phosphodiester linkage or a phosphorothioate linkage;and MO is a modified oligonucleotide, wherein the modifiedoligonucleotide consists of 8 to 22 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary tomiR-21 (SEQ ID NO: 1).

In certain embodiments, a compound has the structureL_(n)-linker-X—N_(m)—Y-MO, wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; X is a phosphodiesterlinkage or a phosphorothioate linkage; Y is a phosphodiester linkage;and MO is a modified oligonucleotide, wherein the modifiedoligonucleotide consists of 8 to 22 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary tomiR-21 (SEQ ID NO: 1).

In certain embodiments, a compound has the structureL_(n)-linker-Y—N_(m)—Y-MO, wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; each Y is a phosphodiesterlinkage; and MO is a modified oligonucleotide, wherein the modifiedoligonucleotide consists of 8 to 22 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary tomiR-21 (SEQ ID NO: 1).

In certain embodiments, if n is greater than 1, L_(n)-linker has thestructure:

wherein each L is, independently, a ligand; n is from 1 to 10; S is ascaffold; and Q′ and Q″ are, independently, linking groups.

In certain embodiments, Q′ and Q″ are each independently selected from apeptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl, asubstituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, a scaffold links 2, 3, 4, or 5 ligands to amodified oligonucleotide. In certain embodiments, a scaffold links 3ligands to a modified oligonucleotide.

In certain embodiments, a compound has the structure:

wherein:

-   -   B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,        —Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—;        -   MO is a modified oligonucleotide, wherein the modified            oligonucleotide consists of 8 to 22 linked nucleosides,            wherein the nucleobase sequence of the modified            oligonucleotide is complementary to miR-21 (SEQ ID NO: 1);        -   R^(N) is selected from H, methyl, ethyl, propyl, isopropyl,            butyl, and benzyl;        -   Z, Z′, and Z″ are each independently selected from O and S;        -   each N is, independently, a modified or unmodified            nucleoside;        -   m is from 1 to 5;        -   X is selected from a phosphodiester linkage and a            phosphorothioate linkage;        -   Y is a phosphodiester linkage; and        -   the wavy line indicates the connection to the rest of the            linker and ligand(s).

In certain embodiments, X is a phosphodiester linkage.

In certain embodiments, n is from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Incertain embodiments, n is 3.

In certain embodiments, at least one ligand is a carbohydrate.

In certain embodiments, at least one ligand is selected from mannose,glucose, galactose, ribose, arabinose, fructose, fucose, xylose,D-mannose, L-mannose, D-galactose, L-galactose, D-glucose, L-glucose,D-ribose, L-ribose, D-arabinose, L-arabinose, D-fructose, L-fructose,D-fucose, L-fucose, D-xylose, L-xylose, alpha-D-mannofuranose,beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose,alpha-D-glucofuranose, Beta-D-glucofuranose, alpha-D-glucopyranose,beta-D-glucopyranose, alpha-D-galactofuranose, beta-D-galactofuranose,alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-ribofuranose,beta-D-ribofuranose, alpha-D-ribopyranose, beta-D-ribopyranose,alpha-D-fructofuranose, alpha-D-fructopyranose, glucosamine,galactosamine, sialic acid, N-acetylgalactosamine.

In certain embodiments, at least one ligand is selected fromN-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, andN-iso-butanoyl-galactosamine.

In certain embodiments, each ligand is N-acetylgalactosamine.

In certain embodiments, a compound has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide, wherein the modified oligonucleotide consistsof 8 to 22 linked nucleosides, wherein the nucleobase sequence of themodified oligonucleotide is complementary to miR-21 (SEQ ID NO: 1).

In certain embodiments, at least one of X₁ and X₂ is a phosphodiesterlinkage. In certain embodiments, each of X₁ and X₂ is a phosphodiesterlinkage.

In certain embodiments, m is 1. In certain embodiments, m is 2. Incertain embodiments, m is 2, 3, 4, or 5. In certain embodiments, m is 3,4, or 5. In certain embodiments, when m is greater than 1, each modifiedor unmodified nucleoside of N_(m) may be connected to adjacent modifiedor unmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or a phosphorothioate internucleoside linkage.

In certain embodiments, N_(m) is N′_(p)N″, wherein each N′ is,independently, a modified or unmodified nucleoside and p is from 0 to 4;and N″ is a nucleoside comprising an unmodified sugar moiety. In certainembodiments, p is 0. In certain embodiments, p is 1, 2, 3, or 4.

In certain embodiments, each N′ comprises an unmodified sugar moiety. Incertain embodiments, each unmodified sugar moiety is, independently, aβ-D-ribose or a β-D-deoxyribose. In certain embodiments, N″ comprises apurine nucleobase. In certain embodiments, at least one N′ comprises apurine nucleobase. In certain embodiments, each purine nucleobase isindependently selected from adenine, guanine, hypoxanthine, xanthine,and 7-methylguanine. In certain embodiments, N″ is aβ-D-deoxyriboadenosine or a β-D-deoxyriboguanosine.

In certain embodiments, where p is 1, 2, 3, or 4, at least one N′comprises a pyrimidine nucleobase. In certain embodiments, N″ comprisesa pyrimidine nucleobase. In certain embodiments, each pyrimidinenucleobase is independently selected from cytosine, 5-methylcytosine,thymine, uracil, and 5,6-dihydrouracil.

In certain embodiments, p is 1, N′ and N″ are each aβ-D-deoxyriboadenosine, and N′ and N″ are linked by a phosphodiesterinternucleoside linkage. In certain embodiments, p is 1, N′ and N″ areeach a β-D-deoxyriboadenosine, and N′ and N″ are linked by aphosphodiester internucleoside linkage. In certain embodiments, p is 1,N′ and N″ are each a β-D-deoxyriboadenosine, and N′ and N″ are linked bya phosphorothioate internucleoside linkage.

In certain embodiments, the sugar moiety of each N is independentlyselected from a β-D-ribose, a β-D-deoxyribose, a 2′-O-methoxy sugar, a2′-O-methyl sugar, a 2′-fluoro sugar, and a bicyclic sugar moiety. Incertain embodiments, each bicyclic sugar moiety is independentlyselected from a cEt sugar moiety, an LNA sugar moiety, and an ENA sugarmoiety. In certain embodiments, a cEt sugar moiety is an S-cEt sugarmoiety. In certain embodiments, a cEt sugar moiety is an R-cEt sugarmoiety.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 22 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1) and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern I in the 5′to 3′ orientation:

(R)_(X)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Q)—N^(Z)

wherein each R is, independently, a non-bicyclic nucleoside; X is from 1to 4; each N^(B) is, independently, a bicyclic nucleoside; each N^(Q)is, independently, a non-bicyclic nucleoside; and each N^(Z) is,independently, a modified nucleoside.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 19 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1) and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern II in the 5′to 3′ orientation:

N^(M)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Q)—N^(Z)

wherein N^(M) is, independently, a modified nucleoside that is not abicyclic nucleoside; each N^(B) is, independently, a bicyclicnucleoside; each N^(Q) is, independently, a non-bicyclic nucleoside; andN^(Z) is, independently, a modified nucleoside.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 19 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1) and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern III in the 5′to 3′ orientation:

(R)_(X)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Y)—N^(Z)

wherein each R is a non-bicyclic nucleoside; X is from 1 to 4; eachN^(B) is a bicyclic nucleoside; each N^(Q) is a non-bicyclic nucleoside;N^(Y) is a modified nucleoside or an unmodified nucleoside; and eachN^(Z) is a modified nucleoside.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 19 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1) and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern IV in the 5′to 3′ orientation:

N^(M)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Y)N^(Z)

wherein N^(M) is a modified nucleoside that is not a bicyclicnucleoside; each N^(B) is a bicyclic nucleoside; each N^(Q) is anon-bicyclic nucleoside; N^(Y) is a modified nucleoside or an unmodifiednucleoside; and N^(Z) is a modified nucleoside.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 19 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1) and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern V in the 5′to 3′ orientation:

N^(M)—N^(B)—(N^(Q)—N^(Q)—N^(B)—N^(B))₄—N^(Z)

wherein N^(M) is a modified nucleoside that is not a bicyclicnucleoside; each N^(B) is a bicyclic nucleoside; each N^(Q) is anon-bicyclic nucleoside; and N^(Z) is a modified nucleoside.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 15 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1), and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern VI in the 5′to 3′ orientation:

N^(Q)—N^(B)—N^(B)—N^(Q)—(N^(B)—N^(B)—N^(Q)—N^(Q))₂—N^(B)—N^(Q)—N^(B)

wherein each N^(Q) is a non-bicyclic nucleoside; and each N^(B) is abicyclic nucleoside.

In any of the embodiments described herein, the modified oligonucleotidemay consist of 8 to 19 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21 (SEQID NO: 1), and wherein the modified oligonucleotide comprises at least 8contiguous nucleosides of the following nucleoside pattern VII in the 5′to 3′ orientation:

N^(M)—(N^(B)—N^(M)—N^(M))₂—N^(M)—(N^(B)—N^(Q)—N^(Q)—N^(Q))₂—N^(B)—N^(B)—N^(Z)

wherein each N^(M) is a modified nucleoside that is not a bicyclicnucleoside; each N^(B) is a bicyclic nucleoside; each N^(Q) is anon-bicyclic nucleoside; and N^(Z) is a modified nucleoside.

In certain embodiments of nucleoside pattern I or III, X is 1, X is 2, Xis 3, or X is 4.

In certain embodiments of any of the compounds provided herein, themodified oligonucleotide comprises at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, or 22contiguous nucleosides of nucleoside pattern I, II, III, IV, or V. Incertain embodiments of any of the compounds provided herein, themodified oligonucleotide comprises at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, or 19 contiguous nucleosides of nucleosidepattern IV, V, or VII. In certain embodiments of any of the compoundsprovided herein, the modified oligonucleotide comprises at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, or 15contiguous nucleosides of nucleoside pattern VI. In certain embodimentsof any of the compounds provided herein, the modified oligonucleotideconsists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22linked nucleosides of nucleoside pattern I, II, III, IV, or V. Incertain embodiments of any of the compounds provided herein, themodified oligonucleotide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, or 19 linked nucleosides of nucleoside pattern IV, V, or VII. Incertain embodiments of any of the compounds provided herein, themodified oligonucleotide consists of 8, 9, 10, 11, 12, 13, 14, or 15linked nucleosides of nucleoside pattern VI.

In certain embodiments of any of the compounds provided herein, thenucleobase sequence of the modified oligonucleotide is at least 90%complementary, is at least 95% complementary, or is 100% complementaryto the nucleobase sequence of miR-21 (SEQ ID NO: 1).

In certain embodiments of any of the compounds provided herein, thenucleobase at position 1 of miR-21 is paired with the first nucleobaseat the 3′-terminus of the modified oligonucleotide.

In certain embodiments of any of the compounds provided herein, eachbicyclic nucleoside is independently selected from an LNA nucleoside, acEt nucleoside, and an ENA nucleoside.

In certain embodiments of any of the compounds provided herein, eachbicyclic nucleoside is a cEt nucleoside. In certain embodiments, the cEtnucleoside is an S-cEt nucleoside. In certain embodiments, the cEtnucleoside is an R-cEt nucleoside. In certain embodiments of any of thecompounds provided herein, each bicyclic nucleoside is an LNAnucleoside.

In certain embodiments of any of the compounds provided herein, eachbicyclic nucleoside comprises a non-methylated nucleobase.

In certain embodiments of any of the compounds provided herein, eachnon-bicyclic nucleoside is independently selected from aβ-D-deoxyribonucleoside, a β-D-ribonucleoside, 2′-O-methyl nucleoside, a2′-O-methoxyethyl nucleoside, and a 2′-fluoronucleoside. In certainembodiments of any of the compounds provided herein, each non-bicyclicnucleoside is independently selected from a β-D-deoxyribonucleoside, anda 2′-O-methoxyethyl nucleoside. In certain embodiments of any of thecompounds provided herein, each non-bicyclic nucleoside is aβ-D-deoxyribonucleoside. In certain embodiments of any of the compoundsprovided herein, each non-bicyclic nucleoside is a 2′-O-methoxyethylnucleoside.

In certain embodiments of any of the compounds provided herein, at leasttwo non-bicyclic nucleosides comprise sugar moieties that are differentfrom one another. In certain embodiments of any of the compoundsprovided herein, each non-bicyclic nucleoside has the same type of sugarmoiety.

In certain embodiments of any of the compounds provided herein, no morethan two non-bicyclic nucleosides are 2′-O-methoxyethyl nucleosides. Incertain such embodiments, each other non-bicyclic nucleoside is aβ-D-deoxyribonucleoside.

In certain embodiments of any of the compounds provided herein, the5′-most and the 3′-most non-bicyclic nucleosides are 2′-O-methoxyethylnucleosides and each other non-bicyclic nucleoside is aβ-D-deoxyribonucleoside. In certain embodiments of any of the compoundsprovided herein, two non-bicyclic nucleosides are 2′-MOE nucleosides andeach other non-bicyclic nucleoside is a β-D-deoxyribonucleoside.

In certain embodiments of nucleoside pattern I or III, each nucleosideof R is a 2′-O-methoxyethyl nucleoside. In certain embodiments ofnucleoside pattern I or III, three nucleosides of R are2′-O-methoxyethyl nucleosides and one nucleoside of R is aβ-D-deoxyribonucleoside.

In certain embodiments of any of the compounds provided herein, at leastone cytosine is a 5-methyl cytosine. In certain embodiments of any ofthe compounds provided herein, each cytosine is a 5-methylcytosine. Incertain embodiments of any of the compounds provided herein, thecytosine at position two of the modified oligonucleotide is a5-methylcytosine.

In certain embodiments of nucleoside pattern I, R consists of fourlinked nucleosides N^(R1)—N^(R2)—N^(R3)—N^(R4), where N^(R1) is a2′-O-methoxyethyl nucleoside and each of N^(R2)—N^(R3)—N^(R4) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments of nucleoside pattern I, each R is a2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEt nucleoside;each N^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a2′-O-methoxyethyl nucleoside. In certain embodiments of nucleosidepattern I, each R is a 2′-O-methoxyethyl nucleoside; X is 1; each N^(B)is an S-cEt nucleoside; each N^(Q) is a 2′-O-methoxyethyl nucleoside;and N^(Z) is a 2′-O-methoxyethyl nucleoside. In certain embodiments ofnucleoside pattern I, each R is a 2′-O-methoxyethyl nucleoside; X is 1;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments of nucleoside pattern I, each R is a 2′-O-methoxyethylnucleoside; X is 1; each N^(B) is an LNA nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethyl nucleoside. Incertain embodiments of nucleoside pattern I, each R is a2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an LNA nucleoside;each N^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is an LNA nucleoside.

In certain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a 2′-O-methoxyethyl nucleoside; and N^(Z) is a2′-O-methoxyethyl nucleoside. In certain embodiments of nucleosidepattern I, N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; each N is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments of nucleoside pattern II, N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an LNA nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethyl nucleoside. Incertain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an LNA nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is an LNA nucleoside.

In certain embodiments of nucleoside pattern III, each R is a2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEt nucleoside;each N^(Q) is a β-D-deoxyribonucleoside; N^(Y) is aβ-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethyl nucleoside. Incertain embodiments of nucleoside pattern III, each R is a2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEt nucleoside;each N^(Q) is a β-D-deoxyribonucleoside; N^(Y) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments of nucleoside pattern III, each R is a 2′-O-methoxyethylnucleoside; X is 1; each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; N^(Y) is an S-cEt nucleoside; and N^(Z) is anS-cEt nucleoside.

In certain embodiments of nucleoside pattern IV, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; N^(Y) is a β-D-deoxyribonucleoside;N^(Z) is a 2′-O-methoxyethyl nucleoside. In certain embodiments ofnucleoside pattern IV, N^(M) is a 2′-O-methoxyethyl nucleoside; eachN^(B) is an S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside;N^(Y) is a β-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. Incertain embodiments of nucleoside pattern IV, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; N^(Y) is an S-cEt nucleoside; andN^(Z) is an S-cEt nucleoside. In certain embodiments of nucleosidepattern IV, N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is independently selected from aβ-D-deoxyribonucleoside and a 2′-O-methoxyethyl nucleoside; N^(Y) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments of nucleoside pattern IV, the modified oligonucleotide hasthe structure:

(SEQ ID NO: 3)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)TA_(S); or(SEQ ID NO: 3)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S).wherein nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; and nucleosides followed by a subscript “S” areS-cEt nucleosides.

In certain embodiments of nucleoside pattern V, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments of nucleoside pattern V, the modifiedoligonucleotide has the structure:

(SEQ ID NO: 3)A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)UsA_(E);wherein nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; and nucleosides followed by a subscript “S” areS-cEt nucleosides.

In certain embodiments of nucleoside pattern VI, each N^(Q) is amodified nucleoside that is not a bicyclic nucleoside. In certainembodiments of nucleoside pattern VI, each N^(Q) is, independently,selected from a 2′-O-methoxyethyl nucleoside and aβ-D-deoxyribonucleoside. In certain embodiments of nucleoside patternVI, each N^(Q) is a 2′-O-methoxyethyl nucleoside. In certain embodimentsof nucleoside pattern VI, each N^(Q) is a β-D-deoxyribonucleoside. Incertain embodiments of nucleoside pattern VI, each N^(Q) is a2′-O-methoxyethyl nucleoside; and each N^(B) is an S-cEt nucleoside. Incertain embodiments of nucleoside pattern VI, each N^(Q) is aβ-D-deoxyribonucleoside nucleoside; and each N^(B) is an S-cEtnucleoside. In any of the embodiments of nucleoside pattern VI, themodified oligonucleotide may have 0, 1, or 2 mismatches with respect tothe nucleobase sequence of miR-21. In certain such embodiments, themodified oligonucleotide has 0 mismatches with respect to the nucleobasesequence of miR-21. In certain embodiments, the modified oligonucleotidehas 1 mismatch with respect to the nucleobase sequence of miR-21. Incertain embodiments, the modified oligonucleotide has 2 mismatches withrespect to the nucleobase sequence of miR-21. In certain embodiments ofnucleoside pattern VI, the modified oligonucleotide has the structure:

(SEQ ID NO: 7)^(Me)C_(E)A_(S)A_(S)T_(E)C_(S)U_(S)A_(E)A_(E)U_(S)A_(S)A_(E)G_(E)C_(S)T_(E)A_(S);wherein nucleosides followed by a subscript “E” are 2′-MOE nucleosides;nucleosides followed by a subscript “S” are S-cEt nucleosides; and^(Me)C is 5-methyl cytosine.

In certain embodiments of nucleoside pattern VII, each N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is independently selected from a 2′-O-methyl nucleoside and aβ-D-deoxyribonucleoside; and N^(Z) is independently selected from anS-cEt nucleoside and a 2′-O-methoxyethyl nucleoside. In certainembodiments of nucleoside pattern VII, each N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments of nucleoside pattern VII, each N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) isindependently selected from a 2′-O-methyl nucleoside and aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments of nucleoside pattern VII, each N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) isindependently selected from a 2′-O-methyl nucleoside and aβ-D-deoxyribonucleoside; and N^(Z) is 2′-O-methoxyethyl nucleoside. Incertain embodiments of nucleoside pattern VII, the modifiedoligonucleotide has the structure:

(SEQ ID NO: 3)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S).

In certain embodiments, a compound comprises a modified oligonucleotideconsisting of 8 to 22 linked nucleosides, wherein the modifiedoligonucleotide comprises at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, or at least 19 contiguous nucleosides of astructure selected from the structures in Table 1. In certainembodiments, a compound comprises a modified oligonucleotide having astructure selected from the structures in Table 1.

In certain embodiments of any of the compounds provided herein, themodified oligonucleotide has the nucleobase sequence of SEQ ID NO: 3,wherein each T in the sequence is independently selected from T and U.In certain embodiments of any of the compounds provided herein, themodified oligonucleotide has the nucleobase sequence of SEQ ID NO: 4,wherein each T in the sequence is independently selected from T and U.

In certain embodiments of any of the compounds provided herein, at leastone internucleoside linkage is a modified internucleoside linkage. Incertain embodiments of any of the compounds provided herein, eachinternucleoside linkage is a modified internucleoside linkage. Incertain embodiments, the modified internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments of any of the compounds provided herein, at leastone nucleoside comprises a modified nucleobase. In certain embodimentsof any of the compounds provided herein, at least one cytosine is a5-methyl cytosine. In certain embodiments of any of the compoundsprovided herein, the modified oligonucleotide has the nucleobasesequence of a sequence selected from SEQ ID NOs: 3 to 10, wherein each Tin the sequence is independently selected from T and U.

In certain embodiments of any of the compounds provided herein, themodified oligonucleotide has 0, 1, 2, or 3 mismatches with respect tothe nucleobase sequence of miR-21. In certain embodiments of any of thecompounds provided herein, the modified oligonucleotide has 0, 1, or 2mismatches with respect to the nucleobase sequence of miR-21. In certainsuch embodiments, the modified oligonucleotide has 0 mismatches withrespect to the nucleobase sequence of miR-21. In certain embodiments,the modified oligonucleotide has 1 mismatch with respect to thenucleobase sequence of miR-21. In certain embodiments, the modifiedoligonucleotide has 2 mismatches with respect to the nucleobase sequenceof miR-21.

Provided herein are methods for inhibiting the activity of miR-21comprising contacting a cell with a compound provided herein. In certainembodiments, the cell is in vivo. In certain embodiments, the cell is invitro. In certain embodiments, the cell is a hepatocyte, a macrophage, amyocyte, an adipocyte, a kupffer cell, a dendritic cell, a fibroblastcell, an epithelial cell, a stellate cell, a keratinocyte, or afibrocyte. In certain embodiments, the cell is a hyperproliferative cellor a hypoxic cell. In certain embodiments, the fibroblast cell is ahyperproliferative fibroblast cell.

Provided herein are methods of inhibiting the activity of miR-21comprising contacting a cell with with any of the compounds describedherein. In certain embodiments, the cell is in vivo. In certainembodiments, the cell is in vitro. In certain embodiments, the cell is afibroblast cell, a hyperproliferative cell, a keratinocyte, or a hypoxiccell.

Provided herein are methods for decreasing collagen expression in a cellcomprising contacting a cell with a compound provided herein.

Provided herein are methods to treat, prevent, or delay the onset of adisease associated with miR-21, comprising administering to a subjecthaving such a disease any of the compounds provided herein.

In certain embodiments, the disease is fibrosis. In certain embodimentsthe fibrosis is kidney fibrosis, lung fibrosis, liver fibrosis, cardiacfibrosis, skin fibrosis, age-related fibrosis, spleen fibrosis,scleroderma, and/or post-transplant fibrosis.

In certain embodiments, the fibrosis is liver fibrosis and is present ina subject having a disease selected from chronic liver injury, hepatitisinfection (such as hepatitis B infection and/or hepatitis C infection),non-alcoholic steatohepatitis, alcoholic liver disease, liver damagefollowing exposure to environmental toxin and/or natural product, andcirrhosis.

Provided herein are methods to treat a fibroproliferative disorder in asubject comprising administering to the subject any of the compoundsprovided herein.

Any of the methods provided herein may comprise selecting a subjecthaving elevated miR-21 expression in one or more tissues.

In certain embodiments, administering any of the compounds providedherein to a subject reduces collagen expression.

In certain embodiments, a subject is in need of improved organ function,wherein the organ function is selected from cardiac function, pulmonaryfunction, liver function, and kidney function. In certain embodiments,the administering of any of the compounds provided herein improves organfunction in the subject, wherein the organ function is selected fromcardiac function, pulmonary function, liver function, and kidneyfunction.

Any of the methods provided herein may comprise evaluating liverfunction in a subject, which may include measuring alanineaminotransferase levels in the blood of the subject; measuring aspartateaminotransferase levels in the blood of the subject; measuring bilirubinlevels in the blood of the subject; measuring albumin levels in theblood of the subject; measuring prothrombin time in the subject;measuring ascites in the subject; measuring encephalopathy in thesubject; and/or measuring liver stiffness, for example, using transientelastography.

Any of the methods provided herein may comprise administering to asubject at least one therapeutic agent selected from ananti-inflammatory agent, an immunosuppressive agent, an anti-diabeticagent, digoxin, a vasodilator, an angiotensin II converting enzyme (ACE)inhibitors, an angiotensin II receptor blockers (ARB), a calcium channelblocker, an isosorbide dinitrate, a hydralazine, a nitrate, ahydralazine, a beta-blocker, a natriuretic peptides, a heparinoid, aconnective tissue growth factor inhibitor, and a transforming growthfactor inhibitor. In certain embodiments, the anti-inflammatory agent isa non-steroidal anti-inflammatory agent, wherein the non-steroidalanti-inflammatory agent is optionally selected from ibuprofen, a COX-1inhibitor and a COX-2 inhibitor. In certain embodiments, theimmunosuppressive agent is selected from a corticosteroid,cyclophosphamide, and mycophenolate mofetil. In certain embodiments,anti-inflammatory agent is a corticosteroid, wherein the corticosteroidis optionally prednisone. In certain embodiments, the angiotensin IIconverting enzyme (ACE) inhibitors is selected from captopril,enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril.In certain embodiments, the angiotensin II receptor blocker (ARB) isselected from candesartan, irbesartan, olmesartan, losartan, valsartan,telmisartan, and eprosartan.

In certain embodiments, a disease is cancer. In certain embodiments, thecancer is liver cancer, breast cancer, bladder cancer, prostate cancer,colon cancer, lung cancer, brain cancer, hematological cancer,pancreatic cancer, head and neck cancer, cancer of the tongue, stomachcancer, skin cancer, thyroid cancer, neuroblastoma, esophageal cancer,mesothelioma, neuroblastoma, bone cancer, kidney cancer, testicularcancer, rectal cancer, cervical cancer, or ovarian cancer. In certainembodiments, the liver cancer is hepatocellular carcinoma. In certainembodiments, the brain cancer is glioblastoma multiforme,oligoastrocytoma, or oligodendroglioma. In certain embodiments, theglioblastoma multiforme is proneural glioblastoma multiforme, neuralglioblastoma multiforme, classical glioblastoma multiforme, ormesenchymal glioblastoma multiforme. In certain embodiments, thehematological cancer is acute myelogenous leukemia, acute lymphocyticleukemia, acute monocytic leukemia, multiple myeloma, chronic lymphoticleukemia, chronic myeloid leukemia, hodgkin's lymphoma, or non-hodgkin'slymphoma. In certain embodiments, the skin cancer is melanoma. Incertain embodiments, the kidney cancer is renal cell carcinoma. Incertain embodiments, the breast cancer is ductal cell carcinoma in situ,invasive ductal cell carcinoma, triple negative breast cancer, medullarycarcinoma, tubular carcinoma, and mucinous carcinoma.

In certain embodiments, the methods provided herein compriseadministering at least one additional anti-cancer therapy to thesubject. In certain embodiments, the anti-cancer therapy is a DNAdamaging agent, a proliferation inhibitor, an anti-folate, a growthfactor receptor inhibitor, an anti-angiogenic agent, a receptor tyrosinekinase inhibitor, a kinase inhibitor, a growth factor inhibitor, acytotoxic agent, radiation therapy, or surgical resection of a tumor. Incertain embodiments, the DNA damaging agent is 1,3-bis(2-chloroethyl)-1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil,cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin,epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin,temozolomide, or topotecan. In certain embodiments, the anti-folate ismethotrexate, aminopterin, thymidylate synthase, serinehydroxymethyltransferase, folyilpolyglutamyl synthetase, g-glutamylhydrolase, glycinamide-ribonucleotide transformylase, leucovorin,amino-imidazole-carboxamide-ribonucleotide transformylase,5-fluorouracil, or a folate transporter. In certain embodiments, thegrowth factor receptor inhibitor is erlotinib, or gefitinib. In certainembodiments, the angiogenesis inhibitor is bevacizumab, thalidomide,carboxyamidotriazole, TNP-470, CM101, IFN-α, platelet factor-4, suramin,SU5416, thrombospondin, a VEGFR antagonist, cartilage-derivedangiogenesis inhibitory factor, a matrix metalloproteinase inhibitor,angiostatin, endostatin, 2-methoxyestradiol, tecogalan,tetrathiomolybdate, prolactin, or linomide. In certain embodiments, thekinase inhibitor is bevacizumab, BIBW 2992, cetuximab, imatinib,trastuzumab, gefitinib, ranibizumab, pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib,E7080, pazopanib, mubritinib, or fostamatinib. In certain embodiments,an anti-cancer agent is anti-miR-21, or anti-miR-221. In certainembodiments, an anti-cancer agent is a miR-34 mimic, including a miR-34amimic, a miR-34b mimic, and/or a miR-34c mimic

In certain embodiments, the administering to a subject having cancerresults in reduction of tumor size and/or tumor number. In certainembodiments, the administering to a subject having cancer prevents ordelays an increase in tumor size and/or tumor number. In certainembodiments, the administering to a subject having cancer prevents orslows metastatic progression. In certain embodiments, the administeringto a subject having cancer extends overall survival time and/orprogression-free survival of the subject. In certain embodiments, themethods provided herein comprise selecting a subject having elevatedserum alpha-fetoprotein and/or elevated serumdes-gamma-carboxyprothrombin. In certain embodiments, the methodsprovided herein comprise reducing serum alpha-fetoprotein and/or serumdes-gamma-carboxyprothrombin. In certain embodiments, the methodsprovided herein comprise selecting an animal having abnormal liverfunction.

In certain embodiments, a subject is a human.

In any of the methods provided herein, the compound is present as apharmaceutical composition.

Any of the compounds provided herein may be for use in therapy. Any ofthe compounds provided herein may be for use in the treatment offibrosis. Any of the compounds provided herein may be for use inpromoting wound healing. Any of the compounds provided herein may be foruse in treating cancer. Any of the compounds provided herein may be foruse in preventing and/or delaying the onset of metastasis.

Any of the compounds provided herein may be for use in the preparationof a medicament. Any of the compounds provided herein may be for use inthe preparation of a medicament for treating fibrosis. Any of thecompounds provided herein may be for use in the preparation of amedicament for promoting wound healing. Any of the compounds providedherein may be for use in the preparation of a medicament for treatingcancer. Any of the compounds provided herein may be for use in thepreparation of a medicament for preventing and/or delaying the onset ofmetastasis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure of a conjugate moiety comprising three GalNAc ligands.

FIG. 2. Conjugated modified oligonucleotide structures.

FIG. 3. Concentration of total compound (A) and unconjugated modifiedoligonucleotide (B) in mouse liver 48 or 168 hours a single subcutaneousinjection of compound 36731 or 40601.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in thearts to which the invention belongs. Unless specific definitions areprovided, the nomenclature utilized in connection with, and theprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well-known and commonly used in the art. In the event thatthere is a plurality of definitions for terms herein, those in thissection prevail. Standard techniques may be used for chemical synthesis,chemical analysis, pharmaceutical preparation, formulation and delivery,and treatment of subjects. Certain such techniques and procedures may befound for example in “Carbohydrate Modifications in Antisense Research”Edited by Sangvi and Cook, American Chemical Society, Washington, D.C.,1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., 18th edition, 1990; and which is hereby incorporated byreference for any purpose. Where permitted, all patents, patentapplications, published applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. Where reference is made to a URL orother such identifier or address, it is understood that such identifierscan change and particular information on the internet can change, butequivalent information can be found by searching the internet. Referencethereto evidences the availability and public dissemination of suchinformation.

Before the present compositions and methods are disclosed and described,it is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

Definitions

“Fibrosis” means the formation or development of excess fibrousconnective tissue in an organ or tissue. In certain embodiments,fibrosis occurs as a reparative or reactive process. In certainembodiments, fibrosis occurs in response to damage or injury. The term“fibrosis” is to be understood as the formation or development of excessfibrous connective tissue in an organ or tissue as a reparative orreactive process, as opposed to a formation of fibrous tissue as anormal constituent of an organ or tissue.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease.

“Subject suspected of having fibrosis” means a subject exhibiting one ormore clinical indicators of fibrosis.

“Fibroproliferative disorder” means a disorder characterized byexcessive proliferation and/or activation of fibroblasts.

“Liver cancer” means a malignant tumor of the liver, either a primarycancer or metastasized cancer. In certain embodiments, liver cancerincludes, but is not limited to, cancer arising from hepatocytes, suchas, for example, hepatomas and hepatocellular carcinomas; fibrolamellarcarcinoma; and cholangiocarcinomas (or bile duct cancer).

“Metastasis” means the process by which cancer spreads from the place atwhich it first arose as a primary tumor to other locations in the body.The metastatic progression of a primary tumor reflects multiple stages,including dissociation from neighboring primary tumor cells, survival inthe circulation, and growth in a secondary location.

“Overall survival time” means the time period for which a subjectsurvives after diagnosis of or treatment for a disease. In certainembodiments, the disease is cancer. In some embodiments, overallsurvival time is survival after diagnosis. In some embodiments, overallsurvival time is survival after the start of treatment.

“Progression-free survival” means the time period for which a subjecthaving a disease survives, without the disease getting worse. In certainembodiments, progression-free survival is assessed by staging or scoringthe disease. In certain embodiments, progression-free survival of asubject having liver cancer is assessed by evaluating tumor size, tumornumber, and/or metastasis.

“Halts further progression” means to stop movement of a medicalcondition to an advanced state.

“Slows further progression” means to reduce the rate at which a medicalcondition moves towards an advanced state.

“Improves life expectancy” means to lengthen the life of a subject bytreating one or more symptoms of a disease in the subject.

“Anti-miR” means an oligonucleotide having a nucleobase sequencecomplementary to a microRNA. In certain embodiments, an anti-miR is amodified oligonucleotide.

“Anti-miR-X” where “miR-X” designates a particular microRNA, means anoligonucleotide having a nucleobase sequence complementary to miR-X. Incertain embodiments, an anti-miR-X is fully complementary to miR-X(i.e., 100% complementary). In certain embodiments, an anti-miR-X is atleast 80%, at least 85%, at least 90%, or at least 95% complementary tomiR-X. In certain embodiments, an anti-miR-X is a modifiedoligonucleotide.

“miR-21” means the mature miRNA having the nucleobase sequence

(SEQ ID NO: 1) UAGCUUAUCAGACUGAUGUUGA.

“miR-21 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence

(SEQ ID NO: 2) UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA.

“Target nucleic acid” means a nucleic acid to which an oligomericcompound is designed to hybridize.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid.

“Target engagement” means the interaction of an oligonucleotide with themicroRNA to which it is complementary, in a manner that changes theactivity, expression or level of the microRNA. In certain embodiments,target engagement means an anti-miR interacting with the microRNA towhich it is complementary, such that the activity of the microRNA isinhibited.

“Modulation” means a perturbation of function, amount, or activity. Incertain embodiments, modulation means an increase in function, amount,or activity. In certain embodiments, modulation means a decrease infunction, amount, or activity.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“5′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 3′-most nucleobase of a particular oligonucleotide.

“3′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 5′-most nucleobase of a particular oligonucleotide.

“Region” means a portion of linked nucleosides within a nucleic acid. Incertain embodiments, an oligonucleotide has a nucleobase sequence thatis complementary to a region of a target nucleic acid. For example, incertain such embodiments an oligonucleotide is complementary to a regionof a microRNA stem-loop sequence. In certain such embodiments, anoligonucleotide is fully complementary to a region of a microRNAstem-loop sequence.

“Segment” means a smaller or sub-portion of a region.

“Nucleobase sequence” means the order of contiguous nucleobases in anoligomeric compound or nucleic acid, typically listed in a 5′ to 3′orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means that one nucleic acid is capable of hybridizing toanother nucleic acid or oligonucleotide. In certain embodiments,complementary refers to an oligonucleotide capable of hybridizing to atarget nucleic acid.

“Fully complementary” means each nucleobase of an oligonucleotide iscapable of pairing with a nucleobase at each corresponding position in atarget nucleic acid. In certain embodiments, an oligonucleotide is fullycomplementary to a microRNA, i.e. each nucleobase of the oligonucleotideis complementary to a nucleobase at a corresponding position in themicroRNA. In certain embodiments, an oligonucleotide wherein eachnucleobase has complementarity to a nucleobase within a region of amicroRNA stem-loop sequence is fully complementary to the microRNAstem-loop sequence.

“Percent complementarity” means the percentage of nucleobases of anoligonucleotide that are complementary to an equal-length portion of atarget nucleic acid. Percent complementarity is calculated by dividingthe number of nucleobases of the oligonucleotide that are complementaryto nucleobases at corresponding positions in the target nucleic acid bythe total number of nucleobases in the oligonucleotide.

“Percent identity” means the number of nucleobases in a first nucleicacid that are identical to nucleobases at corresponding positions in asecond nucleic acid, divided by the total number of nucleobases in thefirst nucleic acid. In certain embodiments, the first nucleic acid is amicroRNA and the second nucleic acid is a microRNA. In certainembodiments, the first nucleic acid is an oligonucleotide and the secondnucleic acid is an oligonucleotide.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of Watson-Crick pairing with a nucleobase at a correspondingposition of a second nucleic acid.

“Identical” in the context of nucleobase sequences, means having thesame nucleobase sequence, independent of sugar, linkage, and/ornucleobase modifications and independent of the methyl state of anypyrimidines present.

“MicroRNA” means an endogenous non-coding RNA between 18 and 25nucleobases in length, which is the product of cleavage of apre-microRNA by the enzyme Dicer. Examples of mature microRNAs are foundin the microRNA database known as miRBase(http://microrna.sanger.ac.uk/). In certain embodiments, microRNA isabbreviated as “microRNA” or “miR.”

“Pre-microRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which is the product of cleavage of a pri-miR by thedouble-stranded RNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature microRNA sequence. Pre-microRNA sequences andstem-loop sequences may overlap. Examples of stem-loop sequences arefound in the microRNA database known as miRBase(http://microrna.sanger.ac.uk/).

“Pri-microRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“microRNA precursor” means a transcript that originates from a genomicDNA and that comprises a non-coding, structured RNA comprising one ormore microRNA sequences. For example, in certain embodiments a microRNAprecursor is a pre-microRNA. In certain embodiments, a microRNAprecursor is a pri-microRNA.

“microRNA-regulated transcript” means a transcript that is regulated bya microRNA. “Monocistronic transcript” means a microRNA precursorcontaining a single microRNA sequence.

“Polycistronic transcript” means a microRNA precursor containing two ormore microRNA sequences.

“Seed sequence” means a nucleobase sequence comprising from 6 to 8contiguous nucleobases of nucleobases 1 to 9 of the 5′-end of a maturemicroRNA sequence.

“Seed match sequence” means a nucleobase sequence that is complementaryto a seed sequence, and is the same length as the seed sequence.

“Oligomeric compound” means a compound that comprises a plurality oflinked monomeric subunits. Oligomeric compounds includedoligonucleotides.

“Oligonucleotide” means a compound comprising a plurality of linkednucleosides, each of which can be modified or unmodified, independentfrom one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar moiety.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“Compound comprising a modified oligonucleotide consisting of” a numberof linked nucleosides means a compound that includes a modifiedoligonucleotide having the specified number of linked nucleosides. Thus,the compound may include additional substituents or conjugates. Unlessotherwise indicated, the compound does not include any additionalnucleosides beyond those of the modified oligonucleotide.

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage. A modified oligonucleotidemay comprise unmodified nucleosides.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified nucleoside” means a nucleoside having any change from anaturally occurring nucleoside. A modified nucleoside may have amodified sugar, and an unmodified nucleobase. A modified nucleoside mayhave a modified sugar and a modified nucleobase. A modified nucleosidemay have a natural sugar and a modified nucleobase. In certainembodiments, a modified nucleoside is a bicyclic nucleoside. In certainembodiments, a modified nucleoside is a non-bicyclic nucleoside.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar moiety” means substitution and/or any change from anatural sugar.

“Unmodified nucleobase” means the naturally occurring heterocyclic basesof RNA or DNA: the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine),and uracil (U).

“5-methylcytosine” means a cytosine comprising a methyl group attachedto the 5 position.

“Non-methylated cytosine” means a cytosine that does not have a methylgroup attached to the 5 position.

“Modified nucleobase” means any nucleobase that is not an unmodifiednucleobase.

“Furanosyl” means a structure comprising a 5-membered ring consisting offour carbon atoms and one oxygen atom.

“Naturally occurring furanosyl” means a ribofuranosyl as found innaturally occurring RNA or a deoxyribofuranosyl as found in naturallyoccurring DNA.

“Sugar moiety” means a naturally occurring furanosyl or a modified sugarmoiety.

“Modified sugar moiety” means a substituted sugar moiety or a sugarsurrogate.

“Substituted sugar moiety” means a furanosyl that is not a naturallyoccurring furanosyl. Substituted sugar moieties include, but are notlimited to sugar moieties comprising modifications at the 2′-position,the 5′-position and/or the 4′-position of a naturally occurringfuranosyl. Certain substituted sugar moieties are bicyclic sugarmoieties.

“Sugar surrogate” means a structure that does not comprise a furanosyland that is capable of replacing the naturally occurring furanosyl of anucleoside, such that the resulting nucleoside is capable of (1)incorporation into an oligonucleotide and (2) hybridization to acomplementary nucleoside. Such structures include relatively simplechanges to the furanosyl, such as rings comprising a different number ofatoms (e.g., 4, 6, or 7-membered rings); replacement of the oxygen ofthe furanosyl with a non-oxygen atom (e.g., carbon, sulfur, ornitrogen); or both a change in the number of atoms and a replacement ofthe oxygen. Such structures may also comprise substitutionscorresponding with those described for substituted sugar moieties (e.g.,6-membered carbocyclic bicyclic sugar surrogates optionally comprisingadditional substituents). Sugar surrogates also include more complexsugar replacements (e.g., the non-ring systems of peptide nucleic acid).Sugar surrogates include without limitation morpholinos, cyclohexenylsand cyclohexitols.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having a 0-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having aO-methoxyethyl modification at the 2′ position.

“2′-O-fluoro” or “2′-F” means a sugar having a fluoro modification ofthe 2′ position. “Bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including by not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl. Nonlimiting exemplary bicyclic sugar moieties includeLNA, ENA, cEt, S-cEt, and R-cEt.

“Locked nucleic acid (LNA) sugar moiety” means a substituted sugarmoiety comprising a (CH₂)—O bridge between the 4′ and 2′ furanose ringatoms.

“ENA sugar moiety” means a substituted sugar moiety comprising a(CH₂)₂—O bridge between the 4′ and 2′ furanose ring atoms.

“Constrained ethyl (cEt) sugar moiety” means a substituted sugar moietycomprising a CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms. In certain embodiments, the CH(CH₃)—O bridge is constrained inthe S orientation. In certain embodiments, the CH(CH₃)—O is constrainedin the R orientation.

“S-cEt sugar moiety” means a substituted sugar moiety comprising anS-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“R-cEt sugar moiety” means a substituted sugar moiety comprising anR-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“2′-O-methyl nucleoside” means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification. A 2′-O-methoxyethyl nucleoside maycomprise a modified or unmodified nucleobase.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification. A 2′-fluoro nucleoside may comprise a modified orunmodified nucleobase.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety. A bicyclic nucleoside may have a modified or unmodifiednucleobase.

“cEt nucleoside” means a nucleoside comprising a cEt sugar moiety. A cEtnucleoside may comprise a modified or unmodified nucleobase.

“S-cEt nucleoside” means a nucleoside comprising an S-cEt sugar moiety.

“R-cEt nucleoside” means a nucleoside comprising an R-cEt sugar moiety.

“Non-bicyclic nucleoside” means a nucleoside that has a sugar other thana bicyclic sugar. In certain embodiments, a non-bicyclic nucleosidecomprises a naturally occurring sugar. In certain embodiments, anon-bicyclic nucleoside comprises a modified sugar. In certainembodiments, a non-bicyclic nucleoside is a β-D-deoxyribonucleoside. Incertain embodiments, a non-bicyclic nucleoside is a 2′-O-methoxyethylnucleoside.

β-D-deoxyribonucleoside” means a naturally occurring DNA nucleoside.β-D-ribonucleoside” means a naturally occurring RNA nucleoside. “LNAnucleoside” means a nucleoside comprising a LNA sugar moiety.

“ENA nucleoside” means a nucleoside comprising an ENA sugar moiety.

“Motif” means a pattern of modified and/or unmodified nucleobases,sugars, and/or internucleoside linkages in an oligonucleotide. Incertain embodiments, a motif is a nucleoside pattern.

“Nucleoside pattern” means a pattern of nucleoside modifications in amodified oligonucleotide or a region thereof. A nucleoside pattern is amotif that describes the arrangement of nucleoside modifications in anoligonucleotide.

“Fully modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage is modified.

“Uniformly modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage has the same modification throughoutthe modified oligonucleotide.

“Stabilizing modification” means a modification to a nucleoside thatprovides enhanced stability to a modified oligonucleotide, in thepresence of nucleases, relative to that provided by 2′-deoxynucleosideslinked by phosphodiester internucleoside linkages. For example, incertain embodiments, a stabilizing modification is a stabilizingnucleoside modification. In certain embodiments, a stabilizingmodification is an internucleoside linkage modification.

“Stabilizing nucleoside” means a nucleoside modified to provide enhancednuclease stability to an oligonucleotide, relative to that provided by a2′-deoxynucleoside. In one embodiment, a stabilizing nucleoside is a2′-modified nucleoside.

“Stabilizing internucleoside linkage” means an internucleoside linkagethat provides improved nuclease stability to an oligonucleotide relativeto that provided by a phosphodiester internucleoside linkage. In oneembodiment, a stabilizing internucleoside linkage is a phosphorothioateinternucleoside linkage.

A “linking group” as used herein refers to an atom or group of atomsthat attach a first chemical entity to a second chemical entity via oneor more covalent bonds.

A “linker” as used herein, refers to an atom or group of atoms thatattach one or more ligands to a modified or unmodified nucleoside viaone or more covalent bonds. The modified or unmodified nucleoside may bepart of a modified oligonucleotide as described herein, or may beattached to a modified oligonucleotide through a phosphodiester orphosphorothioate bond. In some embodiments, the linker attaches one ormore ligands to the 3′ end of a modified oligonucleotide. In someembodiments, the linker attaches one or more ligands to the 5′ end of amodified oligonucleotide. In some embodiments, the linker attaches oneor more ligands to a modified or unmodified nucleoside that is attachedto the 3′ end of a modified oligonucleotide. In some embodiments, thelinker attaches one or more ligands to a modified or unmodifiednucleoside that is attached to the 5′ end of a modified oligonucleotide.When the linker attaches one or more ligands to the 3′ end of a modifiedoligonucleotide or to a modified or unmodified nucleoside attached tothe 3′ end of a modified oligonucleotide, in some embodiments, theattachment point for the linker may be the 3′ carbon of a modified orunmodified sugar moiety. When the linker attaches one or more ligands tothe 5′ end of a modified oligonucleotide or to a modified or unmodifiednucleoside attached to the 5′ end of a modified oligonucleotide, in someembodiments, the attachment point for the linker may be the 5′ carbon ofa modified or unmodified sugar moiety.

“Subject” means a human or non-human animal selected for treatment ortherapy. In certain embodiments, a non-human animal subject is a canine.

“Subject in need thereof” means a subject that is identified as in needof a therapy or treatment.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, andintramuscular administration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Intracardial administration” means administration into the heart. Incertain embodiments, intracardial administration occurs by way of acatheter. In certain embodiments, intracardial administration occurs byway of open heart surgery.

“Pulmonary administration” means administration to the lungs.

“Administered concomitantly” refers to the co-administration of two ormore agents in any manner in which the pharmacological effects of bothare manifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent orpharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments,therapy includes, but is not limited to, chemotherapy, radiationtherapy, or administration of a pharmaceutical agent.

“Treatment” means the application of one or more specific proceduresused for the cure or amelioration of a disease. In certain embodiments,the specific procedure is the administration of one or morepharmaceutical agents.

“Amelioration” means a lessening of severity of at least one indicatorof a condition or disease. In certain embodiments, amelioration includesa delay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“At risk for developing” means the state in which a subject ispredisposed to developing a condition or disease. In certainembodiments, a subject at risk for developing a condition or diseaseexhibits one or more symptoms of the condition or disease, but does notexhibit a sufficient number of symptoms to be diagnosed with thecondition or disease. In certain embodiments, a subject at risk fordeveloping a condition or disease exhibits one or more symptoms of thecondition or disease, but to a lesser extent required to be diagnosedwith the condition or disease.

“Prevent the onset of” means to prevent the development of a conditionor disease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Delay the onset of” means to delay the development of a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Therapeutic agent” means a pharmaceutical agent used for the cure,amelioration or prevention of a disease.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration. In certain embodiments, a dose may beadministered in two or more boluses, tablets, or injections. Forexample, in certain embodiments, where subcutaneous administration isdesired, the desired dose requires a volume not easily accommodated by asingle injection. In such embodiments, two or more injections may beused to achieve the desired dose. In certain embodiments, a dose may beadministered in two or more injections to minimize injection sitereaction in an individual. In certain embodiments, a dose isadministered as a slow infusion.

“Dosage unit” means a form in which a pharmaceutical agent is provided.In certain embodiments, a dosage unit is a vial containing lyophilizedoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted oligonucleotide.

“Therapeutically effective amount” refers to an amount of apharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a sterile aqueoussolution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Improved organ function” means a change in organ function toward normallimits. In certain embodiments, organ function is assessed by measuringmolecules found in a subject's blood or urine. For example, in certainembodiments, improved liver function is measured by a reduction in bloodliver transaminase levels. In certain embodiments, improved kidneyfunction is measured by a reduction in blood urea nitrogen, a reductionin proteinuria, a reduction in albuminuria, etc.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Side effect” means a physiological response attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Suchside effects may be detected directly or indirectly. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Injection site reaction” means inflammation or abnormal redness of skinat a site of injection in an individual.

“Subject compliance” means adherence to a recommended or prescribedtherapy by a subject.

“Comply” means the adherence with a recommended therapy by a subject.

“Recommended therapy” means a treatment recommended by a medicalprofessional to treat, ameliorate, delay, or prevent a disease.

Overview

The activity of a modified oligonucleotide is based on the specifichybridization event that occurs between a modified oligonucleotide andits target RNA and produces a desired pharmacological endpoint. In orderfor this to occur, certain pharmacokinetic processes must take place,for example, delivery of an intact drug to the target cell or tissue,and entry of the modified oligonucleotide into the cell containing thetarget RNA. Modified oligonucleotides may be conjugated to one or moremoieties which improve delivery to the target cell or tissue and/orcellular uptake of the oligonucleotide, ultimately resulting in enhancedpotency. For example, increased cellular uptake of compounds may beachieved by utilizing conjugates that are ligands for cell-surfacereceptors. The binding of a ligand conjugated to an exogenous molecule(e.g., a drug) to its cell surface receptor leads to receptor-mediatedendocytosis of the conjugated molecule, thereby facilitatingtransmembrane transport of the exogenous molecule. For example, thetargeted delivery to hepatocyte cells may be achieved by covalentlyattaching a conjugate comprising a carbohydrate moiety to a modifiedoligonucleotide. Upon recognition and binding of the carbohydrate moietyby the asialoglycoprotein receptor present on the surface of ahepatocyte cell, the conjugated modified oligonucleotide is transportedacross the cell membrane into the hepatocyte. By improving delivery inthis manner, the potency of the modified oligonucleotide can beenhanced, as a lower does of compound is required to achieve the desiredpharmacological endpoint.

Certain conjugates described herein have the advantage of providingimproved delivery to target cell types and also being cleavable in vivoto produce the unconjugated modified oligonucleotide upon in vivoadministration. As described above, in vivo targeting to a specifictissue or cell type may be enhanced by using a conjugate moiety. Oncethe conjugated modified oligonucleotide reaches its site of action,however, the presence of all or part of the covalently-linked conjugatemoiety may alter the activity of certain conjugated modifiedoligonucleotides or may impact the analyses required to understandcertain pharmacokinetic properties of the modified oligonucleotide, suchas half-life in the target cell. As such, it may be desirable toadminister a compound comprising a modified oligonucleotide attached toa conjugate moiety that is sufficiently stable to improve cellularuptake, but also allows for cleavage of the conjugate moiety once thecompound has been internalized by the target cell. Accordingly, providedherein are compounds comprising a modified oligonucleotide linked to acleavable conjugate moiety, which improve the potency of the modifiedoligonucleotide and permit partial or completed release of the modifiedoligonucleotide in its unconjugated form.

miR-21 is a ubiquitously expressed microRNA that is linked to a varietyof cellular processes, including cell differentiation, proliferation,apoptosis and matrix turnover. Additionally, miR-21 is associated withmultiple diseases. miR-21 is frequently upregulated in cancer, andinhibition of miR-21 has demonstrated a reduction in tumor growth inseveral animal models of cancer. Inhibition of miR-21 in an animal modelof cardiac hypertrophy demonstrated a role for miR-21 in heart disease.A role in fibrosis has been demonstrated in animal models of cardiacfibrosis, kidney fibrosis, and lung fibrosis. A study of the inhibitionof miR-21 in a tissue explants model illustrated that the inhibition ofmiR-21 promotes wound healing. As such, inhibitors of miR-21 are usefulin a variety of research and clinical settings.

To identify potent inhibitors of miR-21, a large number of anti-miR-21compounds were designed and synthesized. The compounds varied in length,and in the number, placement, and identity of bicyclic nucleosides andnon-bicyclic nucleosides. An initial series of compounds was tested inan in vitro luciferase assay, which identified a subset of compounds asin vitro active compounds. These in vitro active compounds were thentested in in vivo assays to identify those compounds that are potentinhibitors of miR-21 in vivo. From the initial in vitro and in vivoscreens, certain compounds were selected as the basis for the design ofadditional compounds. The experimentally observed correlations betweenstructure and activity (both in vitro and in vivo) were used to informthe design of these additional compounds, with further variations inlength and selection and arrangement of bicyclic and non-bicyclicnucleosides. Certain compounds were also tested for other properties,for example, susceptibility to exonuclease activity. Of the nearly 300compounds screened in vitro during this process, no more than 50% wereidentified as active in the in vitro luciferase assay. Of these activein vitro compounds, a small subset was identified as both sufficientlystable in the presence of exonucleases, and active in vivo. Through thisiterative process of designing and screening compounds, it was observedthat compounds having particular patterns of bicyclic and non-bicyclicmodifications were stable, potent inhibitors of miR-21 in vivo.

In vivo targeting of the inhibitors of miR-21 to a specific tissue orcell type may be enhanced by using a conjugate moiety described herein.For example, the targeted delivery of the miR-21 inhibitor to hepatocytecells may be achieved by covalently attaching a conjugate comprising acarbohydrate moiety to a modified oligonucleotide. Further, by linkingthe modified oligonucleotide to the conjugate moiety through a cleavablelinker, partial or completed release of the modified oligonucleotide inits unconjugated form may be achieved in the target cell. By improvingdelivery in this manner, the potency of the modified oligonucleotide canbe enhanced, as a lower does of compound is required to achieve thedesired pharmacological endpoint.

As such, these compounds are useful for the modulation of cellularprocesses that are promoted by the activity of miR-21. Further, suchcompounds are useful for treating, preventing, and/or delaying the onsetof diseases associated with miR-21. Such diseases may be characterizedby abnormally high expression of miR-21, relative to non-diseasesamples. Such diseases include, but are not limited to, fibrosis andcancer.

Certain Conjugated Compounds

In certain embodiments, a compound provided herein comprises a conjugatemoiety linked to the 5′ terminus or the 3′ terminus of a modifiedoligonucleotide. In certain embodiments, the compound comprises aconjugate moiety linked to the 3′ terminus of a modifiedoligonucleotide. In certain embodiments, the compound comprises aconjugate moiety linked to the 5′ terminus of a modifiedoligonucleotide. In certain embodiments, the compound comprises a firstconjugate moiety linked to a 3′ terminus of the modified oligonucleotideand a second conjugate moiety linked to the 5′ terminus of a modifiedoligonucleotide.

In certain embodiments, a conjugate moiety comprises at least one ligandselected from a carbohydrate, cholesterol, a lipid, a phospholipid, anantibody, a lipoprotein, a hormone, a peptide, a vitamin, a steroid, ora cationic lipid.

Ligands may be covalently attached to a modified oligonucleotide by anysuitable linker. Various linkers are known in the art, and certainnonlimiting exemplary linkers are described, e.g., in PCT PublicationNo. WO 2013/033230 and U.S. Pat. No. 8,106,022 B2. In some embodiments,a linker may be selected that is resistant to enzymatic cleavage invivo. In some embodiments, a linker may be selected that is resistant tohydrolytic cleavage in vivo. In some embodiments, a linker may beselected that will undergo enzymatic cleavage in vivo. In someembodiments, a linker may be selected that will undergo hydrolyticcleavage in vivo.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure A:

L_(n)-linker-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; and MOis a modified oligonucleotide.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure B:

L_(n)-linker-X₁—N_(m)—X₂-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; each Nis, independently, a modified or unmodified nucleoside and m is from 1to 5; X₁ and X₂ are each, independently, a phosphodiester linkage or aphosphorothioate linkage; and MO is a modified oligonucleotide. Incertain embodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3, 4, or 5. In certain embodiments, m is 2, 3, 4, or5. In certain embodiments, when m is greater than 1, each modified orunmodified nucleoside of N_(m) may be connected to adjacent modified orunmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure C:

L_(n)-linker-X—N_(m)—Y-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; each Nis, independently, a modified or unmodified nucleoside and m is from 1to 5; X is a phosphodiester linkage or a phosphorothioate linkage; Y isa phosphodiester linkage; and MO is a modified oligonucleotide. Incertain embodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3, 4, or 5. In certain embodiments, m is 2, 3, 4, or5. In certain embodiments, when m is greater than 1, each modified orunmodified nucleoside of N_(m) may be connected to adjacent modified orunmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure D:

L_(n)-linker-Y—N_(m)—Y-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; each Nis, independently, a modified or unmodified nucleoside and m is from 1to 5; each Y is a phosphodiester linkage; and MO is a modifiedoligonucleotide. In certain embodiments, m is 1. In certain embodiments,m is 2. In certain embodiments, m is 3, 4, or 5. In certain embodiments,m is 2, 3, 4, or 5. In certain embodiments, when m is greater than 1,each modified or unmodified nucleoside of N_(m) may be connected toadjacent modified or unmodified nucleosides of N_(m) by a phosphodiesterinternucleoside linkage or phosphorothioate internucleoside linkage.

In certain embodiments, when n is greater than 1, the linker comprises ascaffold capable of linking more than one L to the remainder of thecompound (i.e., to the modified oligonucleotide (MO), to X₁—N_(m)—X₂-MO,to X—N_(m)—Y-MO, etc.). In some such embodiments, the L_(n)-linkerportion of the compound (such as a compound of Structure A, B, C, or D)comprises Structure E:

wherein each L is, independently, a ligand; n is from 1 to 10; S is ascaffold; and Q′ and Q″ are, independently, linking groups.

In some embodiments, each Q′ and Q″ is independently selected from apeptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl, asubstituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C_(2o) alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In some embodiments, a scaffold is capable of linking 2, 3, 4, or 5ligands to a modified oligonucleotide. In some embodiments, a scaffoldis capable of linking 3 ligands to a modified oligonucleotide.

A nonlimiting exemplary Structure E is Structure E(i):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, and R₄are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, and R₄ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments, R₁, R₂, R₃, and R₄ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(ii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁ is selected fromH, C₁-C₆ alkyl, and substituted C₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁ is selected from H, methyl, ethyl, propyl,isopropyl, and butyl. In some embodiments, R₁ is H or methyl.

A further nonlimiting exemplary Structure E is Structure E(iii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, R₄, andR₅ are each, independently, selected from H, C₁-C₆ alkyl, andsubstituted C₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, R₄, and R₅ are each,independently, selected from H, methyl, ethyl, propyl, isopropyl, andbutyl. In some embodiments R₁, R₂, R₃, R₄, and R₅ are each selected fromH and methyl.

A further nonlimiting exemplary Structure E is Structure E(iv):

wherein L₁ and L₂ are each, independently, a ligand; Q′₁, Q′₂, and Q″are each, independently, a linking group; and R₁, R₂, and R₃ are each,independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, and Q″ are each, independently, selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid. In someembodiments, R₁, R₂, and R₃ are each, independently, selected from H,methyl, ethyl, propyl, isopropyl, and butyl. In some embodiments R₁, R₂,and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(v):

wherein L₁ and L₂ are each, independently, a ligand; Q′₁, Q′₂, and Q″are each, independently, a linking group; and R₁, R₂, and R₃ are each,independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, and Q″ are each, independently, selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid. In someembodiments, R₁, R₂, and R₃ are each, independently, selected from H,methyl, ethyl, propyl, isopropyl, and butyl. In some embodiments R₁, R₂,and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(vi):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′_(2,)Q′₃, and Q″ are each, independently, a linking group; and R₁, R₂, and R₃are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, and R₃ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(vii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; R₁, R₂, and R₃ areeach, independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl; and Z and Z′ are each independently selected from O and S.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, and R₃ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, and R₃ are each selected from H and methyl. In someembodiments, Z or Z′ on at least one P atom is S, and the other Z or Z′is O (i.e., a phosphorothioate linkage). In some embodiments, each—OP(Z)(Z′)O— is a phosphorothioate linkage. In some embodiments, Z andZ′ are both 0 on at least one P atom (i.e., a phosphodiester linkage).In some embodiments, each —OP(Z)(Z′)O— is a phosphodiester linkage.

A further nonlimiting exemplary Structure E is Structure E(viii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, and R₄are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, and R₄ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, R₃, and R₄ are each selected from H and methyl.

Nonlimiting exemplary scaffolds and/or linkers comprising scaffolds, andsynthesis thereof, are described, e.g., PCT Publication No. WO2013/033230, U.S. Pat. No. 8,106,022 B2, U.S. Publication No.2012/0157509 Al; U.S. Pat. No. 5,994,517; U.S. Pat. No. 7,491,805 B2;U.S. Pat. No. 8,313,772 B2; Manoharan, M., Chapter 16, Antisense DrugTechnology, Crooke, S. T., Marcel Dekker, Inc., 2001, 391-469.

In some embodiments, the L_(n)-linker portion of the compound comprisesStructure F:

wherein:

B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,—Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—;

MO is a modified oligonucleotide;

R^(N) is selected from H, methyl, ethyl, propyl, isopropyl, butyl, andbenzyl;

Z, Z′, and Z″ are each independently selected from O and S;

each N is, independently, a modified or unmodified nucleoside;

m is from 1 to 5;

X is selected from a phosphodiester linkage and a phosphorothioatelinkage;

Y is a phosphodiester linkage; and

the wavy line indicates the connection to the rest of the linker andligand(s).

In certain embodiments, the wavy line indicates a connection toStructure E, above.

In certain embodiments, n is from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Incertain embodiments, n is 1. In certain embodiments, n is 2. In certainembodiments, n is 3. In certain embodiments, n is 4. In certainembodiments, n is 5.

In some embodiments, the L_(n)-linker portion of the compound comprisesStructure G:

wherein:B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,—Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—;

MO is a modified oligonucleotide;

R^(N) is selected from H, methyl, ethyl, propyl, isopropyl, butyl, andbenzyl;

Z, Z′, and Z″ are each independently selected from O and S;

each N is, independently, a modified or unmodified nucleoside;

m is from 1 to 5;

X is selected from a phosphodiester linkage and a phosphorothioatelinkage;

Y is a phosphodiester linkage;

each L is, independently, a ligand; n is from 1 to 10; S is a scaffold;and Q′ and Q″ are, independently, linking groups.

In some embodiments, each Q′ and Q″ are independently selected from apeptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl, asubstituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

A nonlimiting exemplary L_(n)-linker portion (e.g., of Structure F or G)of a compound is shown in Structure H below:

wherein the wavy line indicates attachment to the modifiedoligonucleotide (MO), to X₁, e.g. in Structure B, or to X or Y, e.g., inStructure C, or D.

In certain embodiments, each ligand is a carbohydrate. A compoundcomprising a carbohydrate-conjugated modified oligonucleotide, whenrecognized by a cell surface lectin, is transported across the cellmembrane into the cell. In certain embodiments, a cell surface lectin isa C-type lectin. In certain embodiments, the C-type lectin is present ona Kuppfer cell. In certain embodiments, a C-type lectin is present on amacrophage. In certain embodiments, a C-type lectin is present on anendothelial cell. In certain embodiments, a C-type lectin is present ona monocyte. In certain embodiments, a C-type lectin is present on aleukocyte. In certain embodiments, a C-type lectin is present on adendritic cell. In certain embodiments, a C-type lectin is present on aB cell. A conjugate may facilitate uptake of an anti-miR-21 compoundinto any cell type that expresses a C-type lectin.

In certain embodiments, a C-type lectin is the asialoglycoproteinreceptor (ASGPR). In certain embodiments, a conjugate comprises one ormore ligands having affinity for the ASGPR, including but not limited togalactose or a galactose derivative. In certain embodiments, a ligandhaving affinity for the ASGPR is N-acetylgalactosamine, galactose,galactosamine, N-formylgalactosamine, N-propionyl-galactosamine,N-n-butanoylgalactosamine, or N-iso-butanoyl-galactosamine. Suchconjugates facilitate the uptake of compounds into cells that expressthe ASGPR, for example, hepatocytes and dendritic cells.

In certain embodiments, a ligand is a carbohydrate selected frommannose, glucose, galactose, ribose, arabinose, fructose, fucose,xylose, D-mannose, L-mannose, D-galactose, L-galactose, D-glucose,L-glucose, D-ribose, L-ribose, D-arabinose, L-arabinose, D-fructose,L-fructose, D-fucose, L-fucose, D-xylose, L-xylose,alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose,beta-D-mannopyranose, alpha-D-glucofuranose, Beta-D-glucofuranose,alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-galactofuranose,beta-D-galactofuranose, alpha-D-galactopyranose, beta-D-galactopyranose,alpha-D-ribofuranose, beta-D-ribofuranose, alpha-D-ribopyranose,beta-D-ribopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose,glucosamine, galactosamine, sialic acid, and N-acetylgalactosamine.

In certain embodiments, a ligand is selected from N-acetylgalactosamine,galactose, galactosamine, N-formylgalactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, andN-iso-butanoyl-galactosamine.

In certain embodiments, a ligand is N-acetylgalactosamine.

In certain embodiments, a compound comprises the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide. In certain embodiments, m is 1. In certainembodiments, m is 2. In certain embodiments, m is 3, 4, or 5. In certainembodiments, m is 2, 3, 4, or 5. In certain embodiments, when m isgreater than 1, each modified or unmodified nucleoside of N_(m) may beconnected to adjacent modified or unmodified nucleosides of N_(m) by aphosphodiester internucleoside linkage or phosphorothioateinternucleoside linkage.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage or a phosphorothioate linkage;each N is, independently, a modified or unmodified nucleoside and m isfrom 1 to 5; Y is a phosphodiester linkage; and MO is a modifiedoligonucleotide. In certain embodiments, m is 1. In certain embodiments,m is 2. In certain embodiments, m is 3, 4, or 5. In certain embodiments,m is 2, 3, 4, or 5. In certain embodiments, when m is greater than 1,each modified or unmodified nucleoside of N_(m) may be connected toadjacent modified or unmodified nucleosides of N_(m) by a phosphodiesterinternucleoside linkage or phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage; each N is, independently, amodified or unmodified nucleoside and m is from 1 to 5; Y is aphosphodiester linkage; and MO is a modified oligonucleotide. In certainembodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3, 4, or 5. In certain embodiments, m is 2, 3, 4, or5. In certain embodiments, when m is greater than 1, each modified orunmodified nucleoside of N_(m) may be connected to adjacent modified orunmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or phosphorothioate internucleoside linkage.

In certain embodiments, at least one of X₁ and X₂ is a phosphodiesterlinkage. In certain embodiments, each of X₁ and X₂ is a phosphodiesterlinkage.

In certain embodiments, m is 1. In certain embodiments, m is 2. Incertain embodiments, m is 2, 3, 4, or 5. In certain embodiments, m is 3,4, or 5. In certain embodiments, when m is greater than 1, each modifiedor unmodified nucleoside of N_(m) may be connected to adjacent modifiedor unmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or a phosphorothioate internucleoside linkage. In certainembodiments, when m is 2, the nucleosides of N_(m) are linked by aphosphodiester internucleoside linkage.

In any of the embodiments described herein, N_(m) may be N′_(p)N″, whereeach N′ is, independently, a modified or unmodified nucleoside and p isfrom 0 to 4; and N″ is a nucleoside comprising an unmodified sugarmoiety.

In certain embodiments, p is 0. In certain embodiments, p is 1, 2, 3, or4. In certain embodiments, when p is 1, 2, 3, or 4, each N′ comprises anunmodified sugar moiety.

In certain embodiments, an unmodified sugar moiety is a β-D-ribose or aβ-D-deoxyribose. In certain embodiments, where p is 1, 2, 3, or 4, N′comprises a purine nucleobase. In certain embodiments, N″ comprises apurine nucleobase. In certain embodiments, a purine nucleobase isselected from adenine, guanine, hypoxanthine, xanthine, and7-methylguanine. In certain embodiments, N is a β-D-deoxyriboadenosineor a β-D-deoxyriboguanosine. In certain embodiments, N″ is aβ-D-deoxyriboadenosine or a β-D-deoxyriboguanosine. In some embodiments,p is 1 and N′ and N″ are each a β-D-deoxyriboadenosine.

In certain embodiments, where p is 1, 2, 3, or 4, N′ comprises apyrimidine nucleobase. In certain embodiments, N″ comprises a pyrimidinenucleobase. In certain embodiments, a pyrimidine nucleobase is selectedfrom cytosine, 5-methylcytosine, thymine, uracil, and 5,6-dihydrouracil.

In certain embodiments, the sugar moiety of each N is independentlyselected from a β-D-ribose, a β-D-deoxyribose, a 2′-O-methoxy sugar, a2′-O-methyl sugar, a 2′-fluoro sugar, and a bicyclic sugar moiety. Incertain embodiments, each bicyclic sugar moiety is independentlyselected from a cEt sugar moiety, an LNA sugar moiety, and an ENA sugarmoiety. In certain embodiments, the cEt sugar moiety is an S-cEt sugarmoiety. In certain embodiments, the cEt sugar moiety is an R-cEt sugarmoiety.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is amodified oligonucleotide.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage; m is 2; each N is aβ-D-deoxyriboadenosine; the nucleosides of N are linked by aphosphodiester internucleoside linkage; Y is a phosphodiester linkage;and MO is a modified oligonucleotide.

Additional moieties for conjugation to a modified oligonucleotideinclude phenazine, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, aconjugate group is attached directly to a modified oligonucleotide.

Certain Modified Oligonucleotides Targeted to miR-21

Provided herein are modified oligonucleotides having certain patterns ofbicyclic and non-bicyclic nucleosides. Modified oligonucleotides havingthe patterns identified herein are effective inhibitors of miR-21activity.

Each of the nucleoside patterns illustrated herein is shown in the 5′ to3′ orientation.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 22 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern I in the 5′ to 3′ orientation:

(R)_(X)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Q)—N^(Z)

wherein each R is a non-bicyclic nucleoside; X is from 1 to 4;

each N^(B) is a bicyclic nucleoside;

each N^(Q) is a non-bicyclic nucleoside; and

each N^(Z) is a modified nucleoside.

In certain embodiments of nucleoside pattern I, X is 1. In certainembodiments of nucleoside pattern I, X is 2. In certain embodiments ofnucleoside pattern I, X is 3. In certain embodiments of nucleosidepattern I, X is 4.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 19 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern II in the 5′ to 3′ orientation:

N^(M)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Q)—N^(Z)

wherein N^(M) is a modified nucleoside that is not a bicyclicnucleoside;

each N^(B) is a bicyclic nucleoside;

each N^(Q) is a non-bicyclic nucleoside; and

N^(Z) is a modified nucleoside.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 22 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern III in the 5′ to 3′ orientation:

(R)_(X)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Y)—N^(Z)

wherein each R is a non-bicyclic nucleoside; X is from 1 to 4;

each N^(B) is a bicyclic nucleoside;

each N^(Q) is a non-bicyclic nucleoside;

N^(Y) is a modified nucleoside or an unmodified nucleoside; and

each N^(Z) is a modified nucleoside.

In certain embodiments of nucleoside pattern III, X is 1. In certainembodiments of nucleoside pattern III, X is 2. In certain embodiments ofnucleoside pattern III, X is 3. In certain embodiments of nucleosidepattern III, X is 4.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 19 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern IV in the 5′ to 3′ orientation:

N^(M)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Y)—N^(Z)

wherein N^(M) is a modified nucleoside that is not a bicyclicnucleoside;

each N^(B) is a bicyclic nucleoside;

each N^(Q) is a non-bicyclic nucleoside;

N^(Y) is a modified nucleoside or an unmodified nucleoside; and

N^(Z) is a modified nucleoside.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 19 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern V in the 5′ to 3′ orientation:

N^(M)—N^(B)—(N^(Q)—N^(Q)—N^(B)—N^(B))₄—N^(Z)

wherein N^(M) is a modified nucleoside that is not a bicyclicnucleoside;

each N^(B) is a bicyclic nucleoside;

each N^(Q) is a non-bicyclic nucleoside; and

N^(Z) is a modified nucleoside.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 15 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1), and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern VI in the 5′ to 3′ orientation:

N^(Q)—N^(B)—N^(B)—N^(Q)—(N^(B)—N^(B)—N^(Q)—N^(Q))₂—N^(B)—N^(Q)—N^(B)

wherein each N^(Q) is a non-bicyclic nucleoside; and

each N^(B) is a bicyclic nucleoside.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 8 to 19 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern VII in the 5′ to 3′ orientation:

N^(M)—(N^(B)—N^(M)—N^(M))₂—N^(M)—(N^(B)—N^(Q)—N^(Q)—N^(Q))₂—N^(B)—N^(B)—N^(Z)

wherein each N^(M) is a modified nucleoside that is not a bicyclicnucleoside;

each N^(B) is a bicyclic nucleoside;

each N^(Q) is a non-bicyclic nucleoside; and

N^(Z) is a modified nucleoside.

The following embodiments apply to any of the nucleoside patternsdescribed herein, including nucleoside patterns I to VII.

In certain embodiments, the modified oligonucleotide comprises at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, or 22 contiguous nucleosides of a nucleoside patterndescribed herein.

In certain embodiments of any of the nucleoside patterns describedherein, the nucleobase sequence of the modified oligonucleotide is atleast 90% complementary to miR-21 (SEQ ID NO: 1). In certain embodimentsof any of the nucleoside patterns described herein, the nucleobasesequence of the modified oligonucleotide is at least 95% complementaryto miR-21 (SEQ ID NO: 1). In certain embodiments of any of thenucleoside patterns described herein, the nucleobase sequence of themodified oligonucleotide is 100% complementary to miR-21 (SEQ ID NO: 1).

In certain embodiments of any of the nucleoside patterns describedherein, the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 such that position 1 of the microRNA is pairedwith the 3′-terminal nucleobase of the oligonucleotide. For example:

5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)   ||||||||||||||||||| 3′-ATCGAATAGTCTGACTACA-5′;(an anti-miR-21; SEQ ID NO: 3) 5′-UAGCUUAUCAGACUGAUGUUGA-3′(miR-21; SEQ ID NO: 1)    ||||||||||||||||||||||3′-ATCGAATAGTCTGACTACAACT-5′; (an anti-miR-21; SEQ ID NO: 4)5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)    |||||||||||||||3′-ATCGAATAGTCTGAC-5′; (an anti-miR-21; SEQ ID NO: 5)5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)    ||||||||||||||||3′-ATCGAATAGTCTGACT-5′; (an anti-miR-21; SEQ ID NO: 6)5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)   |||||||||||||||||| 3′-ATCGAATAGTCTGACTAC-5′;(an anti-miR-21; SEQ ID NO: 9)

In certain embodiments of any of the nucleoside patterns describedherein, the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21 such that position 2 of the microRNA is pairedwith the 3′-terminal nucleobase of the oligonucleotide. For example:

5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)   |||||||||||||||||| 3′-TCGAATAGTCTGACTACA-5′;(an anti-miR-21; SEQ ID NO: 10)

In certain embodiments of any of the nucleoside patterns describedherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21, and has 1 to 3 mismatches with respect to thenucleobase sequence of miR-21. In certain embodiments, the modifiedoligonucleotide is complementary to miR-21, and has 1 mismatch withrespect to the nucleobase sequence of miR-21. the modifiedoligonucleotide is complementary to miR-21, and has 2 mismatches withrespect to the nucleobase sequence of miR-21. In certain embodiments,the modified oligonucleotide has the sequence of any one of SEQ ID NOs:3 to 6, 9, and 10, but with 1 or 2 nucleobase changes. For example:

5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)    |||||||| ||| ||3′-ATCGAATAATCTAAC-5′; (an anti-miR-21; SEQ ID NO: 7)5′-UAGCUUAUCAGACUGAUGUUGA-3′ (miR-21; SEQ ID NO: 1)    |||||||||||||||||| 3′-TTCGAATAGTCTGACTACA-5′;(an anti-miR-21; SEQ ID NO: 8)

It is to be understood that, in SEQ ID NOs: 3 to 10, each “T” in thesequence may independently be either a “T” nucleobase or a “U”nucleobase, and that a compound having the sequence of any of SEQ IDNOs: 3 to 10 may comprise all T's, all U's, or any combination of U'sand T's. Thus, the presence of “T” at various positions in SEQ ID NOs: 3to 10 throughout the present disclosure and in the accompanying sequencelisting is not limiting with respect to whether that particularnucleobase is a “T” or a “U.”

In certain embodiments of any of the nucleoside patterns describedherein, each bicyclic nucleoside is independently selected from an LNAnucleoside, a cEt nucleoside, and an ENA nucleoside. In certainembodiments, the sugar moieties of at least two bicyclic nucleosides aredifferent from one another. In certain embodiments, all bicyclicnucleosides have the same sugar moieties as one another. In certainembodiments, each bicyclic nucleoside is a cEt nucleoside. In certainembodiments, each bicyclic nucleoside is an LNA nucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, a cEt nucleoside is an S-cEt nucleoside. In certain embodimentsof any of the nucleoside patterns described herein, a cEt nucleoside isan R-cEt nucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, each non-bicyclic nucleoside is independently selected from aβ-D-deoxyribonucleoside, a β-D-ribonucleoside, a 2′-O-methyl nucleoside,a 2′-O-methoxyethyl nucleoside, and a 2′-fluoronucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, each non-bicyclic nucleoside is independently selected from aβ-D-deoxyribonucleoside, and a 2′-O-methoxyethyl nucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, at least two non-bicyclic nucleosides comprise sugar moietiesthat are different from one another and are independently selected froma β-D-deoxyribonucleoside, a β-D-ribonucleoside, a 2′-O-methylnucleoside, a 2′-O-methoxyethyl nucleoside, and a 2′-fluoronucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, at least two non-bicyclic nucleosides comprise sugar moietiesthat are different from one another and are independently selected froma β-D-deoxyribonucleoside and a 2′-O-methoxyethyl nucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, each non-bicyclic nucleoside is independently selected from aβ-D-deoxyribonucleoside, a 2′-O-methyl nucleoside, and a2′-O-methoxyethyl nucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, each non-bicyclic nucleoside has the same type of sugar moietyand is selected from a β-D-deoxyribonucleoside, a β-D-ribonucleoside, a2′-O-methyl nucleoside, a 2′-O-methoxyethyl nucleoside, and a2′-fluoronucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, each non-bicyclic nucleoside is a β-D-deoxyribonucleoside. Incertain embodiments, each non-bicyclic nucleoside is a 2′-O-methylnucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, no more than 3 of the non-bicyclic nucleosides are2′-O-methoxyethyl nucleosides. In certain embodiments, no more than 2 ofthe non-bicyclic nucleosides are 2′-O-methoxyethyl nucleosides. Incertain embodiments, no more than 1 of the non-bicyclic nucleosides is a2′-O-methoxyethyl nucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, one non-bicyclic nucleoside is a 2′-MOE nucleoside and eachother non-bicyclic nucleosides is a β-D-deoxyribonucleoside. In certainembodiments, two non-bicyclic nucleosides are 2′-O-methoxyethylnucleosides and each other non-bicyclic nucleoside is aβ-D-deoxyribonucleoside. In certain embodiments, three non-bicyclicnucleosides are 2′-O-methoxyethyl nucleosides and each othernon-bicyclic nucleoside is a β-D-deoxyribonucleoside.

In certain embodiments of any of the nucleoside patterns describedherein, the 5′-most non-bicyclic nucleoside and the 3′-most non-bicyclicnucleoside are 2′-O-methoxyethyl nucleosides, and each othernon-bicyclic nucleoside is a β-D-deoxyribonucleoside.

In certain embodiments of any nucleoside pattern I, where X is 4, eachnucleoside of R is a 2′-O-methoxyethyl nucleoside. In certainembodiments of nucleoside pattern I, where X is 4, two nucleosides of Rare β-D-deoxyribonucleosides and two nucleosides of R are2′-O-methoxyethyl nucleosides. In certain embodiments of nucleosidepattern I, where X is 4, three nucleosides of R areβ-D-deoxyribonucleosides and one nucleoside of R is a 2′-O-methoxyethylnucleoside.

In certain embodiments of nucleoside pattern I, R isN^(R1)—N^(R2)—N^(R3)—N^(R4) where N^(R1) is a 2′-MOE nucleoside, N^(R2)is a β-D-deoxyribonucleoside, N^(R3) is a β-D-deoxyribonucleoside, andN^(R4) is a β-D-deoxyribonucleoside.

In certain embodiments of nucleoside pattern I, R consists of fourlinked nucleosides N^(R1)—N^(R2)—N^(R3)—N^(R4), wherein N^(R1) is a2′-O-methoxyethyl nucleoside and each of N^(R2)—N^(R3)—N^(R4) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments, the modified oligonucleotide has thenucleobase sequence of SEQ ID NO: 4, wherein each T in the sequence isindependently selected from T and U.

In certain embodiments of nucleoside pattern I, X is 1 and eachnucleoside of R is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a 2′-O-methoxyethyl nucleoside; andN^(Z) is a 2′-O-methoxyethyl nucleoside. In certain embodiments, themodified oligonucleotide has the nucleobase sequence of SEQ ID NO: 3,wherein each T in the sequence is independently selected from T and U.

In certain embodiments of nucleoside pattern I, X is 4 and eachnucleoside of R is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a 2′-O-methoxyethyl nucleoside; andN^(Z) is a 2′-O-methoxyethyl nucleoside. In certain embodiments, themodified oligonucleotide has the nucleobase sequence of SEQ ID NO: 4,wherein each T in the sequence is independently selected from T and U.

In certain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments, the modified oligonucleotide has thenucleobase sequence of SEQ ID NO: 3, wherein each T in the sequence isindependently selected from T and U.

In certain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside. Incertain embodiments, the modified oligonucleotide has the nucleobasesequence of SEQ ID NO: 3, wherein each T in the sequence isindependently selected from T and U.

In certain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an LNA nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments, the modified oligonucleotide has thenucleobase sequence of SEQ ID NO: 3, wherein each T in the sequence isindependently selected from T and U.

In certain embodiments of nucleoside pattern II, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an LNA nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is an LNA nucleoside. Incertain embodiments, the modified oligonucleotide has the nucleobasesequence of SEQ ID NO: 3, wherein each T in the sequence isindependently selected from T and U.

In certain embodiments of nucleoside pattern III, R is a modifiednucleoside that is not a bicyclic nucleoside; and x is 1. In certainembodiments of nucleoside pattern III, R is a modified nucleoside thatis not a bicyclic nucleoside; x is 1; and each N^(Q) is an unmodifiednucleoside. In certain embodiments of nucleoside pattern III, R is amodified nucleoside that is not a bicyclic nucleoside; x is 1; eachN^(Q) is an unmodified nucleoside; each N^(B) is independently selectedfrom an S-cEt nucleoside and an LNA nucleoside; and N^(Y) is selectedfrom a β-D-deoxyribonucleoside, a 2′-O-methoxyethyl nucleoside, an S-cEtnucleoside, and an LNA nucleoside. In certain embodiments of nucleosidepattern III, R is a modified nucleoside that is not a bicyclicnucleoside; x is 1; and each N^(Q) is a β-D-deoxyribonucleoside. Incertain embodiments of nucleoside pattern III, R is a modifiednucleoside that is not a bicyclic nucleoside; x is 1; each N^(Q) is aβ-D-deoxyribonucleoside; each N^(B) is independently selected from anS-cEt nucleoside and an LNA nucleoside; and N^(Y) is selected from aβ-D-deoxyribonucleoside, a 2′-O-methoxyethyl nucleoside, an S-cEtnucleoside, and an LNA nucleoside. In certain embodiments of nucleosidepattern III, R is a modified nucleoside that is not a bicyclicnucleoside; x is 1; each N^(Q) is an unmodified nucleoside; each N^(B)is an S-cEt nucleoside; and N^(Y) is selected from aβ-D-deoxyribonucleoside, a 2′-O-methoxyethyl nucleoside, and an S-cEtnucleoside. In certain embodiments of nucleoside pattern III, R is amodified nucleoside that is not a bicyclic nucleoside; x is 1; eachN^(Q) is a β-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside;and N^(Y) is selected from a β-D-deoxyribonucleoside, a2′-O-methoxyethyl nucleoside, and an S-cEt nucleoside. In certainembodiments, the modified oligonucleotide of pattern III has anucleobase sequence selected from SEQ ID NOs: 3 to 10, wherein each T inthe sequence is independently selected from T and U.

In certain embodiments of nucleoside pattern IV, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is independently selected froman S-cEt nucleoside and an LNA nucleoside; each N^(Q) is independentlyselected from a β-D-deoxyribonucleoside and a 2′-O-methoxyethylnucleoside; N^(Y) is selected from a 2′-O-methoxyethyl nucleoside, anS-cEt nucleoside, an LNA nucleoside, and a β-D-deoxyribonucleoside; andN^(Z) is selected from a 2′-O-methoxyethyl nucleoside, an LNAnucleoside, and an S-cEt nucleoside. In certain embodiments ofnucleoside pattern IV, N^(M) is a 2′-O-methoxyethyl nucleoside; eachN^(B) is an S-cEt nucleoside; each N^(Q) is independently selected froma β-D-deoxyribonucleoside and a 2′-O-methoxyethyl nucleoside; N^(Y) isselected from a 2′-O-methoxyethyl nucleoside, an S-cEt nucleoside, and aβ-D-deoxyribonucleoside; and N^(Z) is selected from a 2′-O-methoxyethylnucleoside and an S-cEt nucleoside. In certain embodiments of nucleosidepattern IV, N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; N^(Y) is anS-cEt nucleoside; and N^(Z) is an S-cEt nucleoside. In certainembodiments, the modified oligonucleotide of pattern IV has a nucleobasesequence selected from SEQ ID NOs: 3 to 10, wherein each T in thesequence is independently selected from T and U.

In certain embodiments of nucleoside pattern V, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is independently selected froman S-cEt nucleoside and an LNA nucleoside; each N^(Q) is independentlyselected from a β-D-deoxyribonucleoside and a 2′-O-methoxyethylnucleoside; and N^(Z) is selected from a 2′-O-methoxyethyl nucleoside,an LNA nucleoside, and an S-cEt nucleoside. In certain embodiments ofnucleoside pattern V, N^(M) is a 2′-O-methoxyethyl nucleoside; eachN^(B) is an S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside;and N^(Z) is selected from a 2′-O-methoxyethyl nucleoside and an S-cEtnucleoside. In certain embodiments of nucleoside pattern V, N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside. In certain embodiments, the modified oligonucleotide ofpattern V has a nucleobase sequence selected from SEQ ID NOs: 3 to 10,wherein each T in the sequence is independently selected from T and U.

In certain embodiments of nucleoside pattern VI, each N^(B) is an S-cEtnucleoside; and each N^(Q) is a 2′-O-methoxyethyl nucleoside. In certainembodiments of nucleoside pattern VI, each N^(B) is an S-cEt nucleoside;and each N^(Q) is a β-D-deoxyribonucleoside. In certain embodiments, themodified oligonucleotide of pattern VI has a nucleobase sequenceselected from SEQ ID NOs: 5 and 7, wherein each T in the sequence isindependently selected from T and U.

In certain embodiments of nucleoside pattern VII, each N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is independently selected from a 2′-O-methyl nucleoside and aβ-D-deoxyribonucleoside; and N^(Z) is selected from an S-cEt nucleosideand a 2′-O-methoxyethyl nucleoside. In certain embodiments of nucleosidepattern VII, each N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) isan S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and N^(Z)is an S-cEt nucleoside. In certain embodiments of nucleoside patternVII, each N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is independently selected from a2′-O-methyl nucleoside and a β-D-deoxyribonucleoside; and N^(Z) is anS-cEt nucleoside. In certain embodiments of nucleoside pattern VII, eachN^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEtnucleoside; each N^(Q) is independently selected from a 2′-O-methylnucleoside and a β-D-deoxyribonucleoside; and N^(Z) is a2′-O-methoxyethyl nucleoside. In certain embodiments, the modifiedoligonucleotide of pattern VII has a nucleobase sequence selected fromSEQ ID NOs: 3 to 10, wherein each T in the sequence is independentlyselected from T and U.

In certain embodiments, a compound provided herein has at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, or at least 19contiguous nucleosides of a nucleobase sequence and modifications (i.e.,a “structure”) as shown in Table 1. In certain embodiments, a compoundprovided herein has a structure selected from the structures in Table 1.Nucleoside modifications are indicated as follows: nucleosides notfollowed by a subscript indicate β-D-deoxyribonucleosides; nucleosidesfollowed by a subscript “E” indicate 2′-MOE nucleosides; nucleosidesfollowed by a subscript “M” indicate 2′-O-methyl nucleosides;nucleosides followed by a subscript “L” are LNA nucleosides; nucleosidesfollowed by a subscript “S” indicate S-cEt nucleosides. Eachinternucleoside linkage is a phosphorothioate internucleoside linkage.Superscript “Me” indicates a 5-methyl group on the base of thenucleoside.

TABLE 1 Anti-miR-21 compounds SEQ ID Compound #Sequence and Chemistry (5′ to 3′) NO Pattern 25068 T_(E)^(Me)C_(E)A_(E)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)T_(E)G_(E)A_(E)U_(S)A_(E)A_(E)G_(E)C_(S)T_(E)A_(E)4 I, III 25070 A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E) 3I, II, III, IV 25072A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)T_(E)G_(E)A_(E)U_(S)A_(E)A_(E)G_(E)C_(S)T_(E)A_(E)3 I, II, III, IV 25082 T_(E)^(Me)CAAC_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E) 4 I, III 25922 A_(E)^(Me)C_(S)AT_(Me)C_(S)AGT_(Me)C_(S)TGAT_(S)AAG^(Me)C_(s)TA_(E) 3I, II, III, IV 25923 A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(S) 3I, II, III, IV 25924 A_(E)^(Me)C_(S)AT^(Me)C_(S)AGT^(Me)C_(S)TGAT_(S)AAG^(Me)C_(S)TA_(S) 3I, II, III, IV 25114 A_(E)^(Me)C_(L)AT^(Me)C_(L)AGT^(Me)C_(L)TGAT_(L)AAG^(Me)C_(L)TA_(E) 3I, II, III, IV 25115 A_(E)^(Me)C_(L)AT^(Me)C_(L)AGT^(Me)C_(L)TGAT_(L)AAG^(Me)C_(L)TA_(L) 3I, II, III, IV 25221 A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)UsA_(S) 3III, IV 25220A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)U_(S)A_(E) 3 V36328^(Me)C_(E)A_(S)G_(S)T_(E)C_(S)U_(S)G_(E)A_(E)U_(S)A_(S)A_(E)G_(E)C_(S)T_(E)A_(S)5 VI 36284^(Me)C_(E)A_(S)A_(S)T_(E)C_(S)U_(S)A_(E)A_(E)U_(S)A_(S)A_(E)G_(E)C_(S)T_(E)A_(S)7 VI 36232 CA_(S)G_(S)TC_(S)U_(S)GAU_(S)A_(S)AGC_(S)TA_(S) 5 VI 36039A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)TA_(S) 3III, IV 36730 U_(S)CAG_(S)TCU_(S)G_(S)AU_(S)AA_(S)GC_(S)UsA_(S) 6 36731A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S)3 III, IV, VII 36842A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TG_(S)AU_(S)AA_(S)GC_(S)U_(S)A_(S)3 36843A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TG_(M)AU_(S)AA_(M)GC_(S)U_(S)A_(S)3 III, IV, VII 36844A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TG_(M)AU_(S)AA_(M)GC_(S)U_(S)T_(E)8 III, IV, VII 36845A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)A_(S)3 III, IV, VII 36846A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)T_(E)8 III, IV, VII 36847A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)U_(S)T_(E) 8 V36000A_(E)C_(S)ATC_(S)A_(E)G_(E)T_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)A_(S)3 III, IV 36001A_(E)C_(S)ATC_(S)AG_(E)T_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)A_(S) 3III, IV 36002A_(E)C_(S)ATC_(S)AGT_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)A_(S) 3III, IV 36003A_(E)C_(S)ATC_(S)AGTC_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)A_(S) 3 III, IV36004A_(E)C_(S)AT_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)T_(E)8 III, IV 36005A_(E)C_(S)ATC_(S)A_(E)G_(E)T_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)T_(E)8 III, IV 36006A_(E)C_(S)ATC_(S)AG_(E)T_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)T_(E) 8III, IV 36007A_(E)C_(S)ATC_(S)AGT_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)TE 8 III, IV36008 A_(E)C_(S)ATC_(S)AGTC_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)T_(E) 8III, IV 36009A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S) 10III, IV, VII 36010A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)A_(M)AGC_(S)U_(S)10 III, IV, VII 36011A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)AAGC_(S)U_(S)10 III, IV, VII 36012C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 9III, IV, VII 36016A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGA_(M)U_(S)AAGC_(S)U_(S)A_(S)3 III, IV 36017A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGA_(M)U_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36018A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGAU_(S)A_(M)AGC_(S)U_(S)A_(S)3 III, IV 36019A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGAU_(S)A_(M)AGC_(S)U_(S)A_(S) 3III, IV 36020A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(M)A_(S)3 III, IV 36021A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)AAGC_(S)U_(M)A_(S)3 III, IV 36022A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGA_(M)U_(S)AAGC_(S)U_(M)A_(S)3 III, IV 36023A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGA_(M)U_(S)AAGC_(S)U_(M)A_(S) 3III, IV 36024A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)A_(M)AGC_(S)U_(M)A_(S)3 III, IV 36025A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGAU_(S)A_(M)AGC_(S)U_(M)A_(S)3 III, IV 36026A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGAU_(S)A_(M)AGC_(S)U_(M)A_(S) 3III, IV 36027A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(M)A_(S)3 III, IV 36028A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(M)A_(S) 3III, IV 36029A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(M)A_(S) 3III, IV 36030A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(M)T_(E)8 III, IV 36031A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(M)T_(E) 8III, IV 36032A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(M)T_(E) 8III, IV 36033A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(M)AGC_(S)U_(S)A_(S) 3 36034A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(M)GAU_(S)A_(M)AGC_(S)U_(S)A_(S) 3 36035A_(E)C_(S)ATC_(S)A_(M)GTC_(S)U_(M)GAU_(S)A_(M)AGC_(S)U_(S)A_(S) 3III, IV 36040A_(E)C_(S)ATC_(S)A_(S)GTC_(S)T_(E)GAU_(S)A_(E)AGC_(S)U_(S)A_(S) 3 36041A_(E)C_(S)ATC_(S)A_(E)GTC_(S)T_(E)GAU_(S)A_(E)AGC_(S)U_(S)A_(S) 3III, IV 36045A_(E)C_(S)AT_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(SAM)AGC_(S)U_(S)T_(E)8 III, IV 36046A_(E)C_(S)ATC_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)T_(E)8 III, IV 36047A_(E)C_(S)ATC_(S)AG_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)T_(E) 8III, IV 36048A_(E)C_(S)ATC_(S)AGT_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)T_(E) 8III, IV 36049A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(M)AGC_(S)U_(S)T_(E) 8 36050A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(M)GAU_(S)A_(M)AGC_(S)U_(S)T_(E) 8 36051A_(E)C_(S)ATC_(S)A_(M)GTC_(S)U_(M)GAU_(S)A_(M)AGC_(S)U_(S)T_(E) 8III, IV 36055 A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)U_(S)10 V 36239A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(E)AGC_(S)U_(S)A_(S) 3 36968A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36969A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36970A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)T_(E)8 III, IV, VII 36971A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(S)T_(E) 8III, IV 36972A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)TC_(S)TGAU_(S)AAGC_(S)U_(S)T_(E) 8III, IV 36973A_(E)C_(S)AT_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36974A_(E)C_(S)A_(E)TC_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36975A_(E)C_(S)A_(E)T_(E)C_(S)AG_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36976A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)GT_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36977A_(E)C_(S)ATC_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36978A_(E)C_(S)A_(E)T_(E)C_(S)AGT_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36979A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)GTC_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3III, IV 36980A_(E)C_(S)ATC_(S)AG_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3 III, IV36981 A_(E)C_(S)ATC_(S)AGT_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S) 3 III, IV36982A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)T_(E)A_(S)3 III, IV, VII 36984A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAG_(E)C_(S)U_(S)A_(S)3 III, IV, VII 36985A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AA_(E)GC_(S)U_(S)A_(S)3 III, IV, VII 36986A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)A_(E)AGC_(S)U_(S)A_(S)3 III, IV, VII 36988A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(E)U_(S)AAGC_(S)U_(S)A_(S)3 III, IV, VII 36989A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TG_(E)AU_(S)AAGC_(S)U_(S)A_(S)3 III, IV, VII 36990A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)T_(E)GAU_(S)AAGC_(S)U_(S)A_(S)3 III, IV, VII 36992A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)A_(M)AGC_(S)U_(S)A_(S)3 III, IV, VII 36993A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)AAGC_(S)U_(S)A_(S)3 III, IV, VII 36994A_(E)C_(S)AT_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)A_(S)3 III, IV 36995A_(E)C_(S)ATC_(S)A_(E)G_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)A_(S)3 III, IV 36996A_(E)C_(S)ATC_(S)AG_(E)T_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)A_(S) 3III, IV 36997A_(E)C_(S)ATC_(S)AGT_(E)C_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)A_(S) 3III, IV 36998A_(E)C_(S)ATC_(S)AGTC_(S)TGA_(M)U_(S)A_(M)AGC_(S)U_(S)A_(S) 3 III, IV36999A_(E)C_(S)AT_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGA_(E)U_(S)A_(E)AGC_(S)U_(S)A_(S)3 III, IV

In certain embodiments of any of the nucleoside patterns describedherein, a modified oligonucleotide consists of 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or 22 linked nucleosides. In certainembodiments, the modified oligonucleotide comprises at least 8 linkednucleosides of a nucleoside pattern set forth in nucleoside pattern I.In certain embodiments, the modified oligonucleotide comprises at least8 linked nucleosides of a nucleoside pattern set forth in nucleosidepattern II. In certain embodiments, the modified oligonucleotidecomprises at least 8 linked nucleosides of a nucleoside pattern setforth in nucleoside pattern III. In certain embodiments, the modifiedoligonucleotide comprises at least 8 linked nucleosides of a nucleosidepattern set forth in nucleoside pattern IV. In certain embodiments, themodified oligonucleotide comprises at least 8 linked nucleosides of anucleoside pattern set forth in nucleoside pattern V. In certainembodiments, the modified oligonucleotide comprises at least 8 linkednucleosides of a nucleoside pattern set forth in nucleoside pattern IV.In certain embodiments, the modified oligonucleotide comprises at least8 linked nucleosides of a nucleoside pattern set forth in nucleosidepattern VII.

In certain embodiments, a modified oligonucleotide having any of thenucleoside patterns described herein comprises at least one modifiedinternucleoside linkage. In certain embodiments, each internucleosidelinkage is a modified internucleoside linkage. In certain embodiments,the modified internucleoside linkage is a phosphorothioateinternucleoside linkage.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence wherein at least one nucleobase is a cytosine. In certainembodiments, at least one cytosine is a 5-methyl cytosine. In certainembodiments, each cytosine is a 5-methyl cytosine. In certainembodiments, at least one nucleoside comprises a modified nucleobase.

Modified oligonucleotides may undergo cleavage by exonucleases and/orendonucleases at various positions throughout the modifiedoligonucleotide. The products of such enzymatic cleavage may retainmiR-21 inhibitory activity, and as such are considered activemetabolites. As such, a metabolic product of a modified oligonucleotidemay be used in the methods described herein.

In certain embodiments, a modified oligonucleotide targeted to miR-21has a nucleoside pattern selected from Table 2A, where N^(M) is amodified nucleoside that is not a bicyclic nucleoside; each N^(B) is abicyclic nucleoside; each N^(Q) is a non-bicyclic nucleoside; and N^(Z)is a modified nucleoside.

TABLE 2A Metabolic Products of Nucleoside Pattern II 5′ 3′ N^(M) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Z) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Z) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Q) N^(Z) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Z) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Z) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Z) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Z) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Z) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Z) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Z) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Z) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Q) N^(Z) N^(M) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(M) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)

In certain embodiments, a modified oligonucleotide targeted to miR-21has a nucleoside pattern selected from Table 2B, where N^(M) is amodified nucleoside that is not a bicyclic nucleoside; each N^(B) is abicyclic nucleoside; each N^(Q) is a non-bicyclic nucleoside; N^(Y) is amodified nucleoside or an unmodified nucleoside; and N^(Z) is a modifiednucleoside.

TABLE 2B Metabolic Products of Nucleoside Pattern IV 5′ 3′ N^(M) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Y) N^(Z) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(Z) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Y) N^(Z) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(Z) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(Z) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y)N^(Z) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Y) N^(Z) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Y) N^(Z) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y)N^(Z) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(Z) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(Z) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Y) N^(Z) N^(M) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(M) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(B) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Y) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Y) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Y) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Y) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(Y) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Y) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Y)

In certain embodiments, a modified oligonucleotide targeted to miR-21has a nucleoside pattern selected from Table 2C, where N^(M) is amodified nucleoside that is not a bicyclic nucleoside; each N^(B) is abicyclic nucleoside; each N^(Q) is a non-bicyclic nucleoside; and N^(Z)is a modified nucleoside.

TABLE 2C Metabolic Products of Nucleoside Pattern V 5′ 3′ N^(M) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(Q)N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(Q) N^(B) N^(B) N^(Z) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Z) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(B) N^(Z) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(B) N^(Z) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Z) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(Q)N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(Q) N^(B) N^(B) N^(Q)N^(Q) N^(B) N^(B) N^(Z) N^(M) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Y) N^(B) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)

In certain embodiments, a modified oligonucleotide targeted to miR-21has a nucleoside pattern selected from Table 2D, where each N^(B) is abicyclic nucleoside; and each N^(Q) is a non-bicyclic nucleoside.

TABLE 2D Metabolic Products of Nucleoside Pattern VI 5′ 3′ N^(Q) N^(B)N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q)N^(B) N^(B) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(Q) N^(B) N^(Q) N^(B) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q)N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(B) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(B)N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q)N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(B) N^(Q)N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q)N^(B) N^(Q)

In certain embodiments, a modified oligonucleotide targeted to miR-21has a nucleoside pattern selected from Table 2E, where N^(M) is amodified nucleoside that is not a bicyclic nucleoside; each N^(B) is abicyclic nucleoside; each N^(Q) is a non-bicyclic nucleoside; and N^(Z)is a modified nucleoside.

TABLE 2E Metabolic Products of Nucleoside Pattern VII 5′ 3′ N^(M) N^(B)N^(M) N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(B) N^(M) N^(M) N^(B) N^(M) N^(M) N^(M)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(M)N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(B) N^(Z) N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(B) N^(M) N^(M) N^(M)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(M)N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B)N^(Z) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Z) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Z) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B)N^(Z) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(Q)N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(Z) N^(Q) N^(B) N^(Q) N^(Q)N^(Q) N^(B) N^(B) N^(Z) N^(M) N^(B) N^(M) N^(M) N^(B) N^(M) N^(M) N^(M)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(B)N^(M) N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(B) N^(M) N^(M) N^(B)N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(M) N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(M) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(M) N^(B) N^(M) N^(M) N^(M)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(B)N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(B) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(Q) N^(B) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(M) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q)N^(B) N^(B) N^(M) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q)N^(Q) N^(Q) N^(B) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B)N^(Q) N^(Q) N^(B) N^(Q) N^(Q) N^(Q) N^(B) N^(B)

In certain embodiments, a modified oligonucleotide targeted to miR-21has a nucleoside pattern and nucleobase sequence selected from Table 3A,Table 3B, Table 3C, Table 3D, Table 3D, Table 3E, Table 3F, or Table 3G.Nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides. Nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides. Nucleosides followed by a subscript “S”indicate S-cEt nucleosides. Each internucleoside linkage is aphosphorothioate internucleoside linkage. Nucleobases may or may notcomprise a methyl group at the 5′ position.

TABLE 3A Metabolic products of compound # 25070 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ N₁₆ N₁₇ N₁₈ N₁₉ NO A_(E) C_(S) AT C_(S) A G T C_(S) T G A U_(S) A A G C_(S) T A_(E) 3 C_(S) A T C_(S) AG T C_(S) T G A U_(S) A A G C_(S) T A_(E) 9 A T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) T A_(E) 17 T C_(S) A G T C_(S) T G A U_(S) A A G C_(S)T A_(E) 18 C_(S) A G T C_(S) T G A U_(S) A A G C_(S) T A_(E) 19 A G TC_(S) T G A U_(S) A A G C_(S) T A_(E) 20 G T C_(S) T G A U_(S) A A GC_(S) T A_(E) 21 T C_(S) T G A U_(S) A A G C_(S) T A_(E) 22 C_(S) T G AU_(S) A A G C_(S) T A_(E) 23 T G A U_(S) A A G C_(S) T A_(E) 24 G AU_(S) A A G C_(S) T A_(E) A U_(S) A A G C_(S) T A_(E) A_(E) C_(S) A TC_(S) A G T C_(S) T G A U_(S) A A G C_(S) T 25 A_(E) C_(S) A T C_(S) A GT C_(S) T G A U_(S) A A G C_(S) 26 C_(S) A T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) T 27 C_(S) A T C_(S) A G T C_(S) T G A U_(S) A A GC_(S) 28 A T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) T 29 A T C_(S) AG T C_(S) T G A U_(S) A A G C_(S) 30 T C_(S) A G T C_(S) T G A U_(S) A AG C_(S) T 31 T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) 32 C_(S) A G TC_(S) T G A U_(S) A A G C_(S) T 33 C_(S) A G T C_(S) T G A U_(S) A A GC_(S) 34 A G T C_(S) T G A U_(S) A A G C_(S) T 35 A G T C_(S) T G AU_(S) A A G C_(S) 36 G T C_(S) T G A U_(S) A A G C_(S) T 37 G T C_(S) TG A U_(S) A A G C_(S) 38 T C_(S) T G A U_(S) A A G C_(S) T 39 T C_(S) TG A U_(S) A A G C_(S) 40 C_(S) T G A U_(S) A A G C_(S) T 41 C_(S) T G AU_(S) A A G C_(S) T G A U_(S) A A G C_(S) T T G A U_(S) A A G C_(S) G AU_(S) A A G C_(S) T

TABLE 3B Metabolic products of compound # 25923 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ N₁₆ N₁₇ N₁₈ N₁₉ NO A_(E) C_(S) AT C_(S) A G T C_(S) T G A U_(S) A A G C_(S) T A_(S) 3 C_(S) A T C_(S) AG T C_(S) T G A U_(S) A A G C_(S) T A_(S) 9 A T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) T A_(S) 17 T C_(S) A G T C_(S) T G A U_(S) A A G C_(S)T A_(S) 18 C_(S) A G T C_(S) T G A U_(S) A A G C_(S) T A_(S) 19 A G TC_(S) T G A U_(S) A A G C_(S) T A_(S) 20 G T C_(S) T G A U_(S) A A GC_(S) T A_(S) 21 T C_(S) T G A U_(S) A A G C_(S) T A_(S) 22 C_(S) T G AU_(S) A A G C_(S) T A_(S) 23 T G A U_(S) A A G C_(S) T A_(S) 24 G AU_(S) A A G C_(S) T A_(S) A U_(S) A A G C_(S) T A_(S) A_(E) C_(S) A TC_(S) A G T C_(S) T G A U_(S) A A G C_(S) T 25 A_(E) C_(S) A T C_(S) A GT C_(S) T G A U_(S) A A G C_(S) 26 C_(S) A T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) T 27 C_(S) A T C_(S) A G T C_(S) T G A U_(S) A A GC_(S) 28 A T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) T 29 A T C_(S) AG T C_(S) T G A U_(S) A A G C_(S) 30 T C_(S) A G T C_(S) T G A U_(S) A AG C_(S) T 31 T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) 32 C_(S) A G TC_(S) T G A U_(S) A A G C_(S) T 33 C_(S) A G T C_(S) T G A U_(S) A A GC_(S) 34 A G T C_(S) T G A U_(S) A A G C_(S) T 35 A G T C_(S) T G AU_(S) A A G C_(S) 36 G T C_(S) T G A U_(S) A A G C_(S) T 37 G T C_(S) TG A U_(S) A A G C_(S) 38 T C_(S) T G A U_(S) A A G C_(S) T 39 T C_(S) TG A U_(S) A A G C_(S) 40 C_(S) T G A U_(S) A A G C_(S) T 41 C_(S) T G AU_(S) A A G C_(S) T G A U_(S) A A G C_(S) T T G A U_(S) A A G C_(S) G AU_(S) A A G C_(S) T

TABLE 3C Metabolic products of compound # 25221 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ N₁₆ N₁₇ N₁₈ N₁₉ NO A_(E) C_(S) AT C_(S) A G T C_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 3 C_(S) A TC_(S) A G T C_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 9 A T C_(S) A G TC_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 17 T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) U_(S) A_(S) 18 C_(S) A G T C_(S) T G A U_(S) A A GC_(S) U_(S) A_(S) 19 A G T C_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 20G T C_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 21 T C_(S) T G A U_(S) A AG C_(S) U_(S) A_(S) 22 C_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 23 T GA U_(S) A A G C_(S) U_(S) A_(S) 24 G A U_(S) A A G C_(S) U_(S) A_(S) AU_(S) A A G C_(S) U_(S) A_(S) A_(E) C_(S) A T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) U_(S) 25 A_(E) C_(S) A T C_(S) A G T C_(S) T G A U_(S)A A G C_(S) 26 C_(S) A T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) U_(S)27 C_(S) A T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) 28 A T C_(S) A GT C_(S) T G A U_(S) A A G C_(S) U_(S) 29 A T C_(S) A G T C_(S) T G AU_(S) A A G C_(S) 30 T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) U_(S)31 T C_(S) A G T C_(S) T G A U_(S) A A G C_(S) 32 C_(S) A G T C_(S) T GA U_(S) A A G C_(S) U_(S) 33 C_(S) A G T C_(S) T G A U_(S) A A G C_(S)34 A G T C_(S) T G A U_(S) A A G C_(S) U_(S) 35 A G T C_(S) T G A U_(S)A A G C_(S) 36 G T C_(S) T G A U_(S) A A G C_(S) U_(S) 37 G T C_(S) T GA U_(S) A A G C_(S) 38 T C_(S) T G A U_(S) A A G C_(S) U_(S) 39 T C_(S)T G A U_(S) A A G C_(S) 40 C_(S) T G A U_(S) A A G C_(S) U_(S) 41 C_(S)T G A U_(S) A A G C_(S) T G A U_(S) A A G C_(S) U_(S) T G A U_(S) A A GC_(S) G A U_(S) A A G C_(S) U_(S)

TABLE 3D Metabolic products of compound # 25220 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ N₁₆ N₁₇ N₁₈ N₁₉ NO A_(E) C_(S) AT C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) A_(E) 3C_(S) A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S)A_(E) 9 A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S)A_(E) 17 T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S)A_(E) 18 C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S)A_(E) 19 A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) A_(E) 20G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) A_(E) 21 T C_(S) U_(S) GA U_(S) A_(S) A G C_(S) U_(S) A_(E) 22 C_(S) U_(S) G A U_(S) A_(S) A GC_(S) U_(S) A_(E) 23 U_(S) G A U_(S) A_(S) A G C_(S) U_(S) A_(E) 24 G AU_(S) A_(S) A G C_(S) U_(S) A_(E) A U_(S) A_(S) A G C_(S) U_(S) A_(E)A_(E) C_(S) A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S)U_(S) 25 A_(E) C_(S) A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A GC_(S) 26 C_(S) A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S)U_(S) 27 C_(S) A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S)28 A T C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) 29 AT C_(S) A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) 30 T C_(S) A_(S)G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) 31 T C_(S) A_(S) G TC_(S) U_(S) G A U_(S) A_(S) A G C_(S) 32 C_(S) A_(S) G T C_(S) U_(S) G AU_(S) A_(S) A G C_(S) U_(S) 33 C_(S) A_(S) G T C_(S) U_(S) G A U_(S)A_(S) A G C_(S) 34 A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S)35 A_(S) G T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) 36 G T C_(S) U_(S) GA U_(S) A_(S) A G C_(S) U_(S) 37 G T C_(S) U_(S) G A U_(S) A_(S) A GC_(S) 38 T C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) 39 T C_(S) U_(S)G A U_(S) A_(S) A G C_(S) 40 C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S)41 C_(S) U_(S) G A U_(S) A_(S) A G C_(S) U_(S) G A U_(S) A_(S) A G C_(S)U_(S) U_(S) G A U_(S) A_(S) A G C_(S) G A U_(S) A_(S) A G C_(S) U_(S)

TABLE 3E Metabolic products of compound # 36284 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ NO ^(Me)C_(E) A_(S) A_(S) T_(E)C_(S) U_(S) A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) T_(E) A_(S) 7A_(S) A_(S) T_(E) C_(S) U_(S) A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S)T_(E) A_(S) 42 A_(S) T_(E) C_(S) U_(S) A_(E) A_(E) U_(S) A_(S) A_(E)G_(E) C_(S) T_(E) A_(S) 43 T_(E) C_(S) U_(S) A_(E) A_(E) U_(S) A_(S)A_(E) G_(E) C_(S) T_(E) A_(S) 44 C_(S) U_(S) A_(E) A_(E) U_(S) A_(S)A_(E) G_(E) C_(S) T_(E) A_(S) 45 U_(S) A_(E) A_(E) U_(S) A_(S) A_(E)G_(E) C_(S) T_(E) A_(S) 46 A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S)T_(E) A_(S) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) T_(E) A_(S) ^(Me)C_(E)A_(S) A_(S) T_(E) C_(S) U_(S) A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S)T_(E) 47 ^(Me)C_(E) A_(S) A_(S) T_(E) C_(S) U_(S) A_(E) A_(E) U_(S)A_(S) A_(E) G_(E) C_(S) 48 A_(S) A_(S) T_(E) C_(S) U_(S) A_(E) A_(E)U_(S) A_(S) A_(E) G_(E) C_(S) T_(E) 49 A_(S) A_(S) T_(E) C_(S) U_(S)A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) 50 A_(S) T_(E) C_(S) U_(S)A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) T_(E) 51 A_(S) T_(E) C_(S)U_(S) A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) 52 T_(E) C_(S) U_(S)A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) T_(E) 53 T_(E) C_(S) U_(S)A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) 54 C_(S) U_(S) A_(E) A_(E)U_(S) A_(S) A_(E) G_(E) C_(S) T_(E) 55 C_(S) U_(S) A_(E) A_(E) U_(S)A_(S) A_(E) G_(E) C_(S) U_(S) A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S)T_(E) U_(S) A_(E) A_(E) U_(S) A_(S) A_(E) G_(E) C_(S) A_(E) A_(E) U_(S)A_(S) A_(E) G_(E) C_(S) T_(E)

TABLE 3F Metabolic products of compound # 36039 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ N₁₆ N₁₇ N₁₈ N₁₉ NO A_(E) C_(S)A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) TA_(S) 3 C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A AG C_(S) T A_(S) 9 A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S)A A G C_(S) T A_(S) 17 T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) AA G C_(S) T A_(S) 18 C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A GC_(S) T A_(S) 19 A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) T A_(S)20 G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) T A_(S) 21 T_(E) C_(S) T GA U_(S) A A G C_(S) T A_(S) 22 C_(S) T G A U_(S) A A G C_(S) T A_(S) 23T G A U_(S) A A G C_(S) T A_(S) 24 G A U_(S) A A G C_(S) T A_(S) A U_(S)A A G C_(S) T A_(S) A_(E) C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E)C_(S) T G A U_(S) A A G C_(S) T 25 A_(E) C_(S) A_(E) T_(E) C_(S) A_(E)G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) 26 C_(S) A_(E) T_(E) C_(S)A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) T 27 C_(S) A_(E) T_(E)C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) 28 A_(E) T_(E)C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) T 29 A_(E) T_(E)C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) 30 T_(E) C_(S)A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) T 31 T_(E) C_(S) A_(E)G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) 32 C_(S) A_(E) G_(E) T_(E)C_(S) T G A U_(S) A A G C_(S) T 33 C_(S) A_(E) G_(E) T_(E) C_(S) T G AU_(S) A A G C_(S) 34 A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) T35 A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) 36 G_(E) T_(E) C_(S)T G A U_(S) A A G C_(S) T 37 G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S)38 T_(E) C_(S) T G A U_(S) A A G C_(S) T 39 T_(E) C_(S) T G A U_(S) A AG C_(S) 40 C_(S) T G A U_(S) A A G C_(S) T 41 C_(S) T G A U_(S) A A GC_(S) T G A U_(S) A A G C_(S) T T G A U_(S) A A G C_(S) G A U_(S) A A GC_(S) T

TABLE 3G Metabolic products of compound # 36731 SEQ 5′ 3′ ID N₁ N₂ N₃ N₄N₅ N₆ N₇ N₈ N₉ N₁₀ N₁₁ N₁₂ N₁₃ N₁₄ N₁₅ N₁₆ N₁₇ N₁₈ N₁₉ NO A_(E) C_(S)A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) U_(S)A_(S) 3 C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A AG C_(S) U_(S) A_(S) 9 A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G AU_(S) A A G C_(S) U_(S) A_(S) 17 T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T GA U_(S) A A G C_(S) U_(S) A_(S) 18 C_(S) A_(E) G_(E) T_(E) C_(S) T G AU_(S) A A G C_(S) U_(S) A_(S) 19 A_(E) G_(E) T_(E) C_(S) T G A U_(S) A AG C_(S) U_(S) A_(S) 20 G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) U_(S)A_(S) 21 T_(E) C_(S) T G A U_(S) A A G C_(S) U_(S) A_(S) 22 C_(S) T G AU_(S) A A G C_(S) U_(S) A_(S) 23 T G A U_(S) A A G C_(S) U_(S) A_(S) 24G A U_(S) A A G C_(S) U_(S) A_(S) A U_(S) A A G C_(S) U_(S) A_(S) A_(E)C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S)U_(S) 25 A_(E) C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T G AU_(S) A A G C_(S) 26 C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E) C_(S) T GA U_(S) A A G C_(S) U_(S) 27 C_(S) A_(E) T_(E) C_(S) A_(E) G_(E) T_(E)C_(S) T G A U_(S) A A G C_(S) 28 A_(E) T_(E) C_(S) A_(E) G_(E) T_(E)C_(S) T G A U_(S) A A G C_(S) U_(S) 29 A_(E) T_(E) C_(S) A_(E) G_(E)T_(E) C_(S) T G A U_(S) A A G C_(S) 30 T_(E) C_(S) A_(E) G_(E) T_(E)C_(S) T G A U_(S) A A G C_(S) U_(S) 31 T_(E) C_(S) A_(E) G_(E) T_(E)C_(S) T G A U_(S) A A G C_(S) 32 C_(S) A_(E) G_(E) T_(E) C_(S) T G AU_(S) A A G C_(S) U_(S) 33 C_(S) A_(E) G_(E) T_(E) C_(S) T G A U_(S) A AG C_(S) 34 A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) U_(S) 35A_(E) G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S) 36 G_(E) T_(E) C_(S) T GA U_(S) A A G C_(S) U_(S) 37 G_(E) T_(E) C_(S) T G A U_(S) A A G C_(S)38 T_(E) C_(S) T G A U_(S) A A G C_(S) U_(S) 39 T_(E) C_(S) T G A U_(S)A A G C_(S) 40 C_(S) T G A U_(S) A A G C_(S) U_(S) 41 C_(S) T G A U_(S)A A G C_(S) T G A U_(S) A A G C_(S) U_(S) T G A U_(S) A A G C_(S) G AU_(S) A A G C_(S) U_(S)

In certain embodiments, a modified oligonucleotide consists of greaterthan 19 linked nucleosides, and comprises a nucleoside pattern describedherein. The nucleosides that are present in addition to the nucleosidesdescribed by the nucleoside pattern are either modified or unmodified.For example, a modified oligonucleotide consisting of 21 linkednucleosides and having a nucleobase sequence complementary to miR-21 mayhave nucleoside pattern II, IV, V, or VII, which is 19 linkednucleosides in length, or may have nucleoside pattern VI, which is 15nucleosides in length, or may have nucleoside pattern III, which may be19 to 22 nucleosides in length. The additional nucleosides may becomprised of modified or unmodified sugar moieties. In certainembodiments, a modified oligonucleotide consists of 19 linkednucleosides and comprises any of the nucleoside patterns describedherein. In certain embodiments, a modified oligonucleotide consists of20 linked nucleosides and comprises any of the nucleoside patternsdescribed herein. In certain embodiments, a modified oligonucleotideconsists of 21 linked nucleosides and comprises any of the nucleosidepatterns described herein. In certain embodiments, a modifiedoligonucleotide consists of 22 linked nucleosides and comprises any ofthe nucleoside patterns described herein. In certain embodiments, amodified oligonucleotide consists of 23 linked nucleosides and comprisesany of the nucleoside patterns described herein. In certain embodiments,a modified oligonucleotide consists of 24 linked nucleosides andcomprises any of the nucleoside patterns described herein. In certainembodiments, a modified oligonucleotide consists of 25 linkednucleosides and comprises any of the nucleoside patterns describedherein.

Certain Uses of the Invention

Modulation of miR-21 Activity

The compounds provided herein are potent and specific inhibitors ofmiR-21 activity, and are thus useful for modulating miR-21 activity.

MicroRNAs bind to and repress the expression of messenger RNAs. Incertain instances, inhibiting the activity of a microRNA leads tode-repression of the messenger RNA, i.e. the messenger RNA expression isincreased at the level of RNA and/or protein. Provided herein aremethods for modulating the expression of a miR-21-regulated transcript,comprising contacting a cell with a compound of the invention, whereinthe compound comprises a modified oligonucleotide having a sequencecomplementary to a miR-21.

In certain embodiments, a miR-21-regulated transcript is YOD1, andinhibition of miR-21 results in an increase in the level of YOD1 mRNA.In certain embodiments, a miR-21 regulated transcript is PPAR-alpha, andinhibition of miR-21 results in an increase in the level of PPAR-alphamRNA. In certain embodiments, a miR-21-regulated transcript is RNF167.

In certain embodiments, a miR-21-regulated transcript is SPG20. Incertain embodiments, inhibition of miR-21 in the liver results in anincrease in the level of SPG20 mRNA.

In certain embodiments, following contacting a cell with a compound ofthe invention, an at least 1.5-fold increase in the mRNA level of amiR-21-regulated transcript is observed. In certain embodiments,following contacting a cell with a compound of the invention, an atleast 2.0-fold increase in the mRNA level of a miR-21-regulatedtranscript is observed. In certain embodiments, the mRNA level of themicroRNA-regulated transcript increases at least 2.5-fold. In certainembodiments, the mRNA level of the microRNA-regulated transcriptincreases at least 3.0-fold. In certain embodiments, the mRNA level ofthe microRNA-regulated transcript increases at least 3.5-fold. Incertain embodiments, the mRNA level of the microRNA-regulated transcriptincreases at least 4.0-fold. In certain embodiments, the mRNA level ofthe microRNA-regulated transcript increases at least 4.5-fold. Incertain embodiments, the mRNA level of the microRNA-regulated transcriptincreases at least 5.0-fold.

Certain microRNAs are known to target several messenger RNAs, in somecases hundreds of messenger RNAs. Inhibiting the activity of a singlemicroRNA can lead to detectable changes in expression of many of themicroRNAs targets. Provided herein are methods for modulating multiplemiR-21-regulated transcripts, comprising inhibiting the activity ofmiR-21, wherein broad gene expression changes occur.

In certain embodiments, phenotypic changes may be observed followinginhibition of a miR-21 with a compound of the invention. Such phenotypicchanges may occur with or without detectable changes in the expressionof a miR-21-regulated transcript.

Diseases and Disorders

In certain embodiments, provided herein are methods for treatingdiseases associated with miR-21 expression and/or activity, comprisingadministering a compound provided herein. In certain embodiments, amiR-21 associated disease occurs in an organ or tissue that can betargeted by a conjugated modified oligonucleotide provided herein.

In certain embodiments, a miR-21 associated disease occurs in the liver.

In certain embodiments, a miR-21 associated disease occurs in the heart.

In certain embodiments, a miR-21 associated disease occurs in thekidney.

Inhibition of miR-21 in a model of fibrosis leads to decreased collagendeposition and reduced fibrosis. Accordingly, provided herein aremethods for treating, preventing, and/or delaying the onset of fibrosis,comprising administering a compound comprising a modifiedoligonucleotide, wherein the modified oligonucleotide is complementaryto miR-21, to a subject. Also provided herein are compositions fortreating, preventing, and/or delaying the onset of fibrosis, comprisinga compound comprising a modified oligonucleotide, wherein the modifiedoligonucleotide is complementary to miR-21, to a subject. The subjectmay have received a diagnosis of fibrosis, may be at risk for developingfibrosis, or may be suspected of having fibrosis.

In certain embodiments, a subject having fibrosis has kidney fibrosis,lung fibrosis, liver fibrosis, cardiac fibrosis, skin fibrosis,age-related fibrosis, spleen fibrosis, scleroderma, or post-transplantfibrosis.

Many diseases or abnormalities of the kidney are characterized by thepresence of fibrosis. As such, the compounds provided herein are usefulfor treating, ameliorating, preventing, and/or delaying the onset of anykidney disease that is characterized by the presence of fibrosis. Incertain embodiments, a subject having fibrosis has a kidney disease orcondition. In certain embodiments, a subject at risk for developingfibrosis has a kidney disease or condition. In certain embodiments, asubject suspected of having fibrosis has a kidney disease or condition.Accordingly, provided herein are methods for treating a subject having,at risk for developing, or suspected of having fibrosis, wherein thesubject has a kidney disease or condition. The kidney disease orcondition may be one or more of, without limitation, glomerular disease,tubulointerstitial fibrosis, IgA nephropathy, interstitialfibrosis/tubular atrophy, glomerulosclerosis, glomerulonephritis, AlportSyndrome, diabetes mellitus, idiopathic focal segmentalglomerulosclerosis, membranous nephropathy, collapsing glomerulopathy,chronic recurrent kidney infection, diabetes mellitus, diabeticnephropathy, chronic recurrent kidney infection, hypertension, systemichypertension, intraglomerular hypertension, chronic kidney disease,acute or repetitive kidney injury, kidney fibrosis resulting from acuteor chronic exposure to nephrotoxic agents, or end stage renal disease.

Provided herein are methods for improving kidney function in a subject.In certain embodiments, provided herein are methods for delaying and/orpreventing the onset of end stage renal disease. In certain embodiments,provided herein are methods for delaying the need for dialysis in asubject. In certain embodiments, provided herein are methods fordelaying the need for renal transplant in a subject. In certainembodiments, provided herein are methods for delaying impaired kidneyfunction in a subject.

Many diseases or abnormalities of the liver are characterized by thepresence of fibrosis. As such, in certain embodiments, a subject havingfibrosis has a liver disease or condition. In certain embodiments, asubject at risk for developing fibrosis has a liver disease orcondition. In certain embodiments, a subject suspected of havingfibrosis has a liver disease or condition. Accordingly, provided hereinare methods for treating a subject having, at risk for developing, orsuspected of having fibrosis, wherein the subject has a liver disease orcondition. In certain embodiments, a liver disease or condition may beone or more of, without limitation, chronic liver injury, hepatitisvirus infection (including hepatitis C virus infection and hepatitis Bvirus infection), non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD),alcoholic steatohepatitis, bridging fibrosis, or cirrhosis. In certainembodiments a liver disease or condition is associated with exposure totoxic chemicals. In certain embodiments, a liver disease or conditionresults from exposure to pharmaceutical agents, e.g. acetaminophen. Incertain embodiments, a subject receiving chemotherapy is at risk forliver fibrosis and/or chronic liver injury.

The presence or degree of fibrosis may be detected by needle liverbiopsy or through a non-invasive transient elastography method thatevaluates the degree of liver stiffness, such as the FibroScan® method.

Fibrosis may be present in many diseases or abnormalities of the lung.As such, in certain embodiments, a subject having fibrosis has a lungdisease or condition. In certain embodiments, a subject at risk fordeveloping fibrosis has a lung disease or condition. In certainembodiments, a subject suspected of having fibrosis has a lung diseaseor condition. Accordingly, provided herein are methods for treating asubject having, at risk for developing, or suspected of having fibrosis,wherein the subject has a lung disease or condition. In certainembodiments, a lung disease or condition may be one or more of, withoutlimitation, lung fibrosis, idiopathic pulmonary fibrosis, or chronicobstructive lung disease. In certain embodiments, lung fibrosis mayresult from inhalation of particulate matter, such as those found insilica gel, asbestos, air pollutants or cigarette smoke.

In certain embodiments the fibrosis is cardiac fibrosis.

In certain embodiments the fibrosis is skin fibrosis. In certainembodiments the fibrosis is age-related fibrosis. In certain embodimentsthe fibrosis is spleen fibrosis. In certain embodiments, the fibrosis isscleroderma.

Fibrosis frequently occurs in transplanted organs, leading to loss oforgan function and ultimately to chronic rejection of the transplantedorgan. Prevention or treatment of fibrosis in transplanted organs mayprevent or delay chronic rejection of the transplanted organ, or inother words may prolong function of the transplanted organ. Accordingly,in certain embodiments a subject has post-transplant fibrosis. Incertain embodiments, the post-transplant fibrosis is kidneypost-transplant fibrosis. In certain embodiments, the transplantationassociated fibrosis is liver post-transplant fibrosis. In certainembodiments, a compound described herein is administered prior totransplantation. In certain embodiments, a compound described herein isadministered concurrently with transplantation. In certain embodiments,a compound described herein is administered following transplantation.

Provided herein are methods for treating a subject having afibroproliferative disorder. In certain embodiments such methodscomprise administering to a subject having or suspected of having afibroproliferative disorder a modified oligonucleotide having anucleobase sequence which is complementary to a miRNA or a precursorthereof. In certain embodiments, the miRNA is miR-21.

Cancer and Metastasis

Abnormally high expression of miR-21 has been demonstrated in numeroustypes of cancer. Further, inhibition of miR-21 in in vitro and in vivomodels has demonstrated that inhibitors of miR-21 are useful for theinhibition of cellular processes that support cancer cell growth, aswell as for the treatment of cancer.

Accordingly, in certain embodiments, the compounds provided herein areused for treating, preventing, ameliorating, and/or delaying the onsetof cancer. In certain embodiments, the cancer is liver cancer, breastcancer, bladder cancer, prostate cancer, bone cancer, colon cancer, lungcancer, brain cancer, hematological cancer, pancreatic cancer, head andneck cancer, cancer of the tongue, stomach cancer, skin cancer, thyroidcancer, neuroblastoma, esophageal cancer, mesothelioma, neuroblastoma,kidney cancer, testicular cancer, rectal cancer, cervical cancer, orovarian cancer. In certain embodiments, the liver cancer ishepatocellular carcinoma. In certain embodiments, the liver cancer isdue to metastasis of cancer that originated in another part of the body,for example a cancer that is due to metastasis of bone cancer, coloncancer or breast cancer. In certain embodiments, the brain cancer isglioblastoma multiforme, oligoastrocytoma, or oligodendroglioma. Incertain embodiments, the glioblastoma multiforme is proneuralglioblastoma multiforme, neural glioblastoma multiforme, classicalglioblastoma multiforme, or mesenchymal glioblastoma multiforme. Incertain embodiments, In certain embodiments, the hematological cancer isacute myelogenous leukemia, acute lymphocytic leukemia, acute monocyticleukemia, multiple myeloma, chronic lymphotic leukemia, chronic myeloidleukemia, hodgkin's lymphoma, or non-hodgkin's lymphoma. In certainembodiments, the skin cancer is melanoma. In certain embodiments, thekidney cancer is renal cell carcinoma. In certain embodiments, thebreast cancer is ductal cell carcinoma in situ, invasive ductal cellcarcinoma, triple negative breast cancer, medullary carcinoma, tubularcarcinoma, and mucinous carcinoma. In certain embodiments, the cancer isresistant to chemotherapy.

In certain embodiments, in liver cancer, miR-21 is elevated and thelevel of one or more miR-21-regulated transcripts is reduced. In certainembodiments, the reduced miR-21-regulated transcript is SPG20.

In certain embodiments, the liver cancer is hepatocellular carcinoma(HCC). The diagnosis of hepatocellular carcinoma is typically made byliver imaging tests such as abdominal ultrasound, helical computedtomography (CT) scan or triple phase CT scan. Such imaging tests may beperformed in conjunction with measurement of blood levels ofalpha-fetoprotein and/or blood levels of des-gamma-carboxyprothrombin.In certain subjects, MRI may be used in place of CT scan. The liverimaging tests allow the assessment of the tumor size, number, location,metastasis outside the liver, patency and or invasion of the arteriesand veins of the liver by the tumor. This assessment aids the decisionas to the mode of therapeutic or palliative intervention that isappropriate. The final diagnosis is typically confirmed by needle biopsyand histopathological examination.

Accordingly, in certain embodiments, the liver cancer is detectedfollowing a computed tomography (CT) scan that detects tumors. Incertain embodiments, the liver cancer is detected following magneticresonance imaging (MRI). In certain embodiments, HCC is characterized asa single primary tumor. In certain embodiments, HCC is characterized asmultiple primary tumors. In certain embodiments, HCC is characterized asa poorly defined primary tumor with an infiltrative growth pattern. Incertain embodiments, the HCC is a single primary tumor with vascularinvasion. In certain embodiments, the HCC is characterized as multipleprimary tumors with vascular invasion. In certain embodiments, the HCChas metastasized to one or more lymph nodes. In certain suchembodiments, the lymph nodes are regional lymph nodes. In certainembodiments, the HCC has metastasized to one or more distant tissues. Incertain embodiments, the HCC has metastasized to other regions of theliver, the portal vein, lymph nodes, adrenal glands, bone or lungs. Incertain embodiments, fibrosis is present.

A number of systems have been employed to predict the prognosis for HCC,including the TNM system, the Okuda system, the Barcelona Clinic LiverCancer (BCLC) and the CLIP score. Each of these systems incorporatesfour features that have been recognized as being important determinantsof survival: the severity of underlying liver disease, the size of thetumor, extension of the tumor into adjacent structures, and the presenceof metastases. The TNM system classifies HCC as stage I, II, III, IV, orV. The BCLC classifies HCC as Stage A1, A2, A3, A4, B, C, and D, andincludes consideration of a Child-Pugh score.

In certain embodiments, liver cancer is classified as Stage 1, Stage 2,Stage 3A, Stage 3B, Stage 3C, or Stage 4. Stage 1 is characterized by acancer is no bigger than 2 cm in size and that has not begun to spread.At Stage 2, the cancer is affecting blood vessels in the liver, or thereis more than one tumor in the liver. At Stage 3A, the cancer is biggerthan 5 cm in size or has spread to the blood vessels near the liver. AtStage 3B, the cancer has spread to nearby organs, such as the bowel orthe stomach, but has not spread to the lymph nodes. At Stage 3C thecancer can be of any size and has spread to nearby lymph nodes. At Stage4 the cancer has spread to parts of the body further away from theliver, such as the lungs.

Biomarkers in a subject's blood may be used to augment a diagnosis ofliver cancer, stage a liver cancer, or develop a prognosis for survival.Such biomarkers include blood tumor biomarkers, such asalpha-fetoprotein and des-gamma carboxyprothrombin. In certain suchembodiments, the subject has elevated blood alpha-fetoprotein. Incertain such embodiments, the subject has elevated blood des-gammacarboxyprothrombin.

A subject having liver cancer may also suffer from abnormal liverfunction. Liver function may be assessed by liver function tests, whichmeasure, among other things, blood levels of liver transaminases. Incertain embodiments, a subject having abnormal liver function haselevated blood liver transaminases. Blood liver transaminases includealanine aminotransferase (ALT) and aspartate aminotransferase (AST). Incertain embodiments, a subject having abnormal liver function haselevated blood bilirubin. In certain embodiments, a subject has abnormalblood albumin levels.

In certain embodiments, a subject's liver function is assessed by theChild-Pugh classification system, which defines three classes of liverfunction. In this classification system, points are assigned tomeasurements in one of five categories: bilirubin levels, albuminlevels, prothrombin time, ascites, and encephalopathy. One point isassigned per each of the following characteristics present: bloodbilirubin of less than 2.0 mg/dl; blood albumin of greater than 3.5mg/dl; a prothrombin time of less than 1.7 international normalizedratio (INR); ascites is absent; or encephalopathy is absent. Two pointsare assigned per each of the following characteristics present: bloodbilirubin of 2-3 mg/dl; blood bilirubin of 3.5 to 2.8 mg/dl; prothrombintime of 1.7-2.3 INR; ascites is mild to moderate; or encephalopathy ismild. Three points are assigned per each of the followingcharacteristics present: bilirubin of greater than 3.0 mg/dl; bloodalbumin of less than 2.8 mg/dl; prothrombin time of greater than 2.3INR; ascites is severe to refractory; or encephalopathy is severe. Thescores are added and Class A is assigned for a score of 5-6 points,Class B is assigned for a score of 7-9 points, and Class C is assignedfor a score of 10-15 points,

A subject having liver cancer may have previously suffered from, or maycurrently suffer from, chronic hepatitis C infection, chronic hepatitisB infection, non-alcoholic fatty liver disease, or cirrhosis. Subjectshaving liver cancer accompanied by and/or resulting from hepatitis Cinfection, hepatitis B infection, non-alcoholic fatty liver disease, orcirrhosis may be treated by the methods described herein.

A subject's response to treatment may be evaluated by tests similar tothose used to diagnosis the liver cancer, including, without limitation,CT scan, MRI, and needle biopsy. Response to treatment may also beassessed by measuring biomarkers in blood, for comparison topre-treatment levels of biomarkers.

miR-21 has also been linked to the process of metastasis. Whileepithelial-mesenchymal transition (EMT) occurs in normal physiologicalprocesses, EMT has been connected to the process of metastasis. Therelevance of EMT in tumor progression has been explored in severalstudies (Greenburg, G. and Hay, E. 1986. Dev. Biol. 115: 363-379; Boyer,B. et al., 1989. J. Cell. Biol. 109: 1495-1509; Uehara, Y. et al., 1992.J. Cell. Biol. 117: 889-894). Epithelial cells are held together throughintegrins to an underlying extracellular matrix (ECM) called thebasement membrane. Mesenchymal cells, on the other hand, have theability to invade and move through the three-dimensional structure ofthe ECM. Therefore, EMT at least superficially resembles thetransformation of normal adherent cells into the metastatic phenotype.

Provided herein are methods for treating, preventing, ameliorating,and/or delaying the onset of metastasis. The metastasis may result fromthe migration of cancer cells from any primary site of cancer to anysecondary site of cancer.

Acute Kidney Injury

Acute kidney injury is a rapid loss of kidney function, which may bebrought on by a number of causes, including low blood volume, exposureto toxins, and urinary obstruction. Acute kidney injury may progress tofibrosis and/or chronic kidney disease. Elevated miR-21 has beenobserved in a model of acute kidney injury. Accordingly, in certainembodiments, the compounds provided herein are used for treating,preventing, ameliorating, and/or delaying fibrosis that occurs as aresult of of acute kidney injury. In certain embodiments, acute kidneyinjury may be the result of exposure to toxic substances, such asenvironmental toxins or cancer therapeutic agents. Acute kidney injurymay arise from damage to the kidney itself, for example in conditionssuch as glomerulonephritis, acute tubular necrosis, and acuteinterstitial nephritis. In certain embodiments, acute kidney injury iscaused by urinary tract obstruction, such as that related to benignprostatic hyperplasia, kidney stones, obstructed urinary catheter,bladder stone, bladder, ureteral or renal malignancy. In someembodiments, the compounds provided herein are administered to a subjectto enhance recovery from acute kidney injury.

Cardiac Diseases

Elevated miR-21 expression has been found in human cardiac disease, andinhibition of miR-21 in relevant animal models has demonstratedimprovements in cardiac fibrosis and cardiac function. Accordingly, incertain embodiments, the compounds provided herein are used fortreating, preventing, ameliorating, and/or delaying the onset of onemore cardiac diseases. In certain embodiments, a cardiac disease iscardiac fibrosis, cardiac enlargement, cardiac hypertrophy, cardiacdilation, hypertrophic cardiomyopathy, heart failure, post-myocardialinfarction remodeling, myocardial infarction, cardiomyopathy (forexample, hypertrophic cardiomyopathy, restrictive cardiomyopathy,dilated cardiomyopathy (DCM), idiopathic dilated cardiomyopathy, ordilated cardiomyopathy with arrhythmias), diastolic heart failure,chronic atrial fibrillation, primary pulmonary hypertension, acuterespiratory distress syndrome, brugada syndrome, progressive cardiacconduction disease, uremic pericarditis, anthracycline cardiomyopathy,arterial fibrosis, post-radiation lymphatic fibrosis, sarcoidosis,scleroderma, endocardial fibroelastosis, serotonergic excess, cardiacvalvulopathy, atrial fibrosis, atrial fibrillation, mitral valvulardisease, hypertension, chronic ventricular dysfunction, pressure andvolume overload, or myocardial fibrosis.

Cellular Processes

Provided herein are compositions and methods for reducing or preventingfibroblast proliferation or activation. Also provided herein arecompositions and methods for inhibiting the synthesis of extracellularmatrix, which includes but is not limited to the synthesis of collagen,fibronectin, collagenase, or a tissue inhibitor of metalloproteinase.

Provided herein are methods for modulating the cellular processesassociated with epithelial-mesenchymal transition (EMT). Such methodscomprise contacting an epithelial cell with a compound consisting of amodified oligonucleotide, wherein the modified oligonucleotide iscomplementary to miR-21. In certain embodiments, the contacting delaysthe transition of an epithelial cell to a fibroblast. In certainembodiments, the contacting prevents the transition of an epithelialcell to a fibroblast.

In certain embodiments, a compound provided herein may stop, slow, orreduce the proliferation of cancer cells. In certain embodiments, acompound provided herein may induce apoptosis in cancer cells. Incertain embodiments, a compound provided herein may reduce cancer cellsurvival.

In certain embodiments, the epithelial cell is a cancer cell. In certainembodiments, the contacting delays the metastasis of the cancer cell. Incertain embodiments, the contacting prevents metastasis of the cancercell.

Certain Clinical Outcomes

In certain embodiments, administration of the compounds or methodsprovided herein result in one or more clinically desirable outcomes in asubject. Such improvements may be used to determine the extent to whicha subject is responding to treatment.

In certain embodiments a clinically desirable outcome is theamelioration of fibrosis. In certain embodiments a clinically desirableoutcome is the slowing of further progression of fibrosis. In certainembodiments a clinically desirable outcome is the halting of furtherprogression of fibrosis. In certain embodiments a clinically desirableoutcome is a reduction in fibrosis. In certain embodiments a clinicallydesirable outcome is a reduction in collagen content in the organ havingfibrosis.

In certain embodiments a clinically desirable outcome is theamelioration of fibrosis in any organ or tissue. In certain embodimentsa clinically desirable outcome is the slowing of further progression offibrosis. In certain embodiments a clinically desirable outcome is thehalting of further progression of fibrosis. In certain embodiments aclinically desirable outcome is a reduction in fibrosis. In certainembodiments a clinically desirable outcome is a reduction in collagencontent in the affected organ.

In certain embodiments a clinically desirable outcome is improved kidneyfunction. In any of the embodiments provided herein, the administrationof a modified oligonucleotide targeted to miR-21 improves one or moremarkers of kidney function in the subject. Improvements in markers ofkidney function include, without limitation: reduced blood urea nitrogenin the subject; reduced creatinine in the blood of the subject; improvedcreatinine clearance in the subject; reduced proteinuria in the subject;reduced albumin:creatinine ratio in the subject; improved glomerularfiltration rate in the subject; improved inulin clearance in thesubject; reduced neutrophil gelatinase-associated lipocalin (NGAL) inthe blood of the subject; reduced Cystatin C in the blood of thesubject; and increased urinary output in the subject. In certainembodiments, the proteinuria is microalbuminuria. In certainembodiments, the proteinuria is macroalbuminuria.

In certain embodiments, the administration delays time to dialysis. Incertain embodiments, the administration delays time to renal transplant.In certain embodiments, the administration improves life expectancy ofthe subject. In certain embodiments, the administration reduceshematuria. In certain embodiments, the administration delays the onsetof hematuria. In certain embodiments, the administration reducesproteinuria. In certain embodiments, the administration delays the onsetof proteinuria.

In certain embodiments, a clinically desirable outcome is improved liverfunction. Liver function may be assessed by one or more known methodscommonly performed in a clinical setting, including, without limitation:measuring alanine aminotransferase levels in the blood of the subject;measuring aspartate aminotransferase levels in the blood of the subject;measuring bilirubin levels in the blood of the subject; measuringalbumin levels in the blood of the subject; measuring prothrombin timein the subject; measuring ascites in the subject; and/or measuringencephalopathy in the subject.

In certain embodiments a clinically desirable outcome is improved lungfunction in a subject having pulmonary fibrosis. In certain embodimentsthe subject has idiopathic pulmonary fibrosis. Lung function may beassessed by one or more known methods commonly performed in a clinicalsetting, including, without limitation: measuring vital capacity in thesubject; measuring forced vital capacity in the subject; measuringforced expiratory volume in one second in the subject; measuring peakexpiratory flow rate in the subject; measuring forced expiratory flow inthe subject; measuring maximal voluntary ventilation in the subject;determining the ratio of forced expiratory volume in one second toforced vital capacity in the subject; measuring ventilation/perfusionratio in the subject; measuring nitrogen washout in the subject;measuring absolute volume of air in one or more lungs of a subject; andadministering the 6-minute walk test.

In certain embodiments a clinically desirable outcome is improvedcardiac function in a subject having cardiac fibrosis. Cardiac functionmay be assessed by one or more known methods commonly performed in aclinical setting, including, without limitation: measuring cardiacoutput in the subject; measuring stroke volume in the subject; measuringmean systolic ejection rate in the subject; measuring systolic bloodpressure in the subject; measuring left ventricular ejection fraction inthe subject; determining stroke index in the subject; determiningcardiac index in the subject; measuring left ventricular percentfractional shortening in the subject; measuring mean velocity ofcircumferential fiber shortening in the subject; measuring leftventricular inflow velocity pattern in the subject; measuring pulmonaryvenous flow velocity pattern in the subject; measuring peak earlydiastolic velocity of the mitral annulus of the subject.

In certain embodiments a clinically desirable outcome is reduction oftumor number and/or reduction of tumor size in a subject having cancer.In certain embodiments a clinically desirable outcome is a reduction incancer cell number in a subject having cancer. Additional clinicallydesirable outcomes include the extension of overall survival time of thesubject, and/or extension of progression-free survival time of thesubject. In certain embodiments, administration of a compound providedherein prevents an increase in tumor size and/or tumor number. Incertain embodiments, administration of a compound provided hereinprevents metastatic progression. In certain embodiments, administrationof a compound provided herein slows or stops metastatic progression. Incertain embodiments, administration of a compound provided hereinprevents the recurrence of tumors. In certain embodiments,administration of a compound provided herein prevents recurrence oftumor metastasis.

Certain desirable clinical outcomes may be assessed by measurements ofblood biomarkers. In certain embodiments, administration of a compoundprovided herein may result in the decrease of blood alpha-fetoproteinand/or blood des-gamma carboxyprothrombin. Administration of a compoundprovided herein may further result in the improvement of liver function,as evidenced by a reduction in blood ALT and/or AST levels.

Certain Additional Therapies

Treatments for fibrosis or any of the conditions listed herein maycomprise more than one therapy. As such, in certain embodiments providedherein are methods for treating a subject having or suspected of havingfibrosis comprising administering at least one therapy in addition toadministering a modified oligonucleotide having a nucleobase sequencecomplementary to a miR-21.

In certain embodiments, the at least one additional therapy comprises apharmaceutical agent.

In certain embodiments, pharmaceutical agents include anti-inflammatoryagents. In certain embodiments, an anti-inflammatory agent is asteroidal anti-inflammatory agent. In certain embodiments, a steroidanti-inflammatory agent is a corticosteroid. In certain embodiments, acorticosteroid is prednisone. In certain embodiments, ananti-inflammatory agent is a non-steroidal anti-inflammatory drug. Incertain embodiments, a non-steroidal anti-inflammatory agent isibuprofen, a COX-I inhibitor, or a COX-2 inhibitor.

In certain embodiments, pharmaceutical agents include immunosuppressiveagents. In certain embodiments, an immunosuppressive agent is acorticosteroid, cyclophosphamide, or mycophenolate mofetil.

In certain embodiments, pharmaceutical agents include anti-diabeticagents. Antidiabetic agents include, but are not limited to, biguanides,glucosidase inhibitors, insulins, sulfonylureas, thiazolidenediones,GLP-1 analogs, and DPP-IV inhibitors.

In certain embodiments, pharmaceutical agents include angiotensin IIreceptor blockers (ARB). In certain embodiments, an angiotensin IIreceptor blocker is candesartan, irbesartan, olmesartan, losartan,valsartan, telmisartan, or eprosartan.

In certain embodiments, pharmaceutical agents include angiotensin IIconverting enzyme (ACE) inhibitors. In certain embodiments, an ACEinhibitor is captopril, enalapril, lisinopril, bnazepril, quinapril,fosinopril, or ramipril.

In certain embodiments, an additional therapy is dialysis. In certainembodiments, an additional therapy is renal transplant.

In certain embodiments, pharmaceutical agents include, but are notlimited to, diuretics (e.g. sprionolactone, eplerenone, furosemide),inotropes (e.g. dobutamine, milrinone), digoxin, vasodilators, calciumchannel blockers, isosorbide dinitrate, hydralazine, nitrates (e.g.isosorbide mononitrate, isosorbide dinitrate), hydralazine,beta-blockers (e.g. carvedilol, metoprolol), and natriuretic peptides(e.g. nesiritide).

In certain embodiments, pharmaceutical agents include heparinoids. Incertain embodiments, a heparinoid is pentosan polysulfate.

In certain embodiments, a pharmaceutical agent is a pharmaceutical agentthat blocks one or more responses to fibrogenic signals.

In certain embodiments, a pharmaceutical agent is an anti-connectivetissue growth factor therapy. In certain embodiments, an anti-CTGFtherapy is a monoclonal antibody against CTGF. In certain embodiments, apharmaceutical agent is an anti-transforming growth factor β therapy. Incertain embodiments, an anti-TGF-β therapy is a monoclonal antibodyagainst TGF-β.

In certain embodiments, an additional therapy may be a pharmaceuticalagent that enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In certain embodiments, the additional therapy is selected to treat orameliorate a side effect of one or more pharmaceutical compositions ofthe present invention. Such side effects include, without limitation,injection site reactions, liver function test abnormalities, renalfunction abnormalities, liver toxicity, renal toxicity, central nervoussystem abnormalities, and myopathies. For example, increasedaminotransferase levels in serum may indicate liver toxicity or liverfunction abnormality. For example, increased bilirubin may indicateliver toxicity or liver function abnormality.

Further examples of additional pharmaceutical agents include, but arenot limited to, immunoglobulins, including, but not limited tointravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen);salicylates; antibiotics; antivirals; antifungal agents; adrenergicmodifiers; hormones (e.g., anabolic steroids, androgen, estrogen,calcitonin, progestin, somatostatin, and thyroid hormones);immunomodulators; muscle relaxants; antihistamines; osteoporosis agents(e.g., biphosphonates, calcitonin, and estrogens); prostaglandins,antineoplastic agents; psychotherapeutic agents; sedatives; poison oakor poison sumac products; antibodies; and vaccines.

Cancer treatments often comprise more than one therapy. As such, incertain embodiments the present invention provides methods for reducingor preventing metastasis comprising administering to a subject acompound comprising a modified oligonucleotide, wherein the modifiedoligonucleotide is complementary to miR-21, and administering at leastone additional therapy that is an anti-cancer therapy.

In certain embodiments, an anti-cancer therapy is chemotherapy. Suitablechemotherapeutic agents include docetaxel, cyclophosphamide, ifosfamide,methotrexate, vinblastine, cisplatin, 5-fluorouracil, gemcitabine,doxorubicin, mitomycin c, sorafenib, etoposide, carboplatin, epirubicin,irinotecan and oxaliplatin. An additional suitable chemotherapeuticagent includes an oligomeric compound, other than a composition targetedto miR-21 provided herein, that is used to treat cancer.

In certain embodiments, an anti-cancer therapy is radiation therapy. Incertain embodiments, an anti-cancer therapy is surgical resection of atumor. In certain embodiments, an anti-cancer therapy is a DNA damagingagent, a proliferation inhibitor, an anti-folate, a growth factorreceptor inhibitor, an anti-angiogenic agent, a receptor tyrosine kinaseinhibitor, a kinase inhibitor, a growth factor inhibitor, or a cytotoxicagent.

In certain embodiments, a DNA damaging agent is 1,3-bis(2-chloroethyl)-1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil,cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin,epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin,temozolomide, or topotecan.

In certain embodiments, an anti-folate is methotrexate, aminopterin,thymidylate synthase, serine hydroxymethyltransferase,folyilpolyglutamyl synthetase, g-glutamyl hydrolase,glycinamide-ribonucleotide transformylase, leucovorin,amino-imidazole-carboxamide-ribonucleotide transformylase,5-fluorouracil, or a folate transporter.

In certain embodiments, a growth factor receptor is erlotinib, orgefitinib. In certain embodiments, an angiogenesis inhibitor isbevacizumab, thalidomide, carboxyamidotriazole, TNP-470, CM101, IFN-α,platelet factor-4, suramin, SU5416, thrombospondin, a VEGFR antagonist,cartilage-derived angiogenesis inhibitory factor, a matrixmetalloproteinase inhibitor, angiostatin, endostatin,2-methoxyestradiol, tecogalan, tetrathiomolybdate, prolactin, orlinomide.

In certain embodiments, a kinase inhibitor is bevacizumab, BIBW 2992,cetuximab, imatinib, trastuzumab, gefitinib, ranibizumab, pegaptanib,sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib,panitumumab, vandetanib, E7080, pazopanib, mubritinib, or fostamatinib.

Certain MicroRNA Nucleobase Sequences

The modified oligonucleotides having a nucleoside pattern describedherein have a nucleobase sequence that is complementary to miR-21 (SEQID NO: 1), or a precursor thereof (SEQ ID NO: 2). In certainembodiments, each nucleobase of the modified oligonucleotide is capableof undergoing base-pairing with a nucleobase at each correspondingposition in the nucleobase sequence of miR-21. In certain embodimentsthe nucleobase sequence of a modified oligonucleotide may have one ormore mismatched base pairs with respect to the nucleobase sequence ofmiR-21 or precursor sequence, and remains capable of hybridizing to itstarget sequence.

As the miR-21 sequence is contained within the miR-21 precursorsequence, a modified oligonucleotide having a nucleobase sequencecomplementary to miR-21 is also complementary to a region of the miR-21precursor.

In certain embodiments, a modified oligonucleotide consists of a numberof linked nucleosides that is equal to the length of miR-21.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of miR-21. A modifiedoligonucleotide having a number of linked nucleosides that is less thanthe length of miR-21, wherein each nucleobase of the modifiedoligonucleotide is complementary to each nucleobase at a correspondingposition of miR-21, is considered to be a modified oligonucleotidehaving a nucleobase sequence that is fully complementary to a region ofthe miR-21 sequence. For example, a modified oligonucleotide consistingof 19 linked nucleosides, where each nucleobase is complementary to acorresponding position of miR-21 that is 22 nucleobases in length, isfully complementary to a 19 nucleobase region of miR-21. Such a modifiedoligonucleotide has 100% complementarity to a 19 nucleobase portion ofmiR-21, and is considered to be 100% complementary to miR-21.

In certain embodiments, a modified oligonucleotide comprises anucleobase sequence that is complementary to a seed sequence, i.e. amodified oligonucleotide comprises a seed-match sequence. In certainembodiments, a seed sequence is a hexamer seed sequence. In certain suchembodiments, a seed sequence is nucleobases 1-6 of miR-21. In certainsuch embodiments, a seed sequence is nucleobases 2-7 of miR-21. Incertain such embodiments, a seed sequence is nucleobases 3-8 of miR-21.In certain embodiments, a seed sequence is a heptamer seed sequence. Incertain such embodiments, a heptamer seed sequence is nucleobases 1-7 ofmiR-21. In certain such embodiments, a heptamer seed sequence isnucleobases 2-8 of miR-21. In certain embodiments, the seed sequence isan octamer seed sequence. In certain such embodiments, an octamer seedsequence is nucleobases 1-8 of miR-21. In certain embodiments, anoctamer seed sequence is nucleobases 2-9 of miR-21.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence having one mismatch with respect to the nucleobase sequence ofmiR-21, or a precursor thereof. In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence having two mismatches withrespect to the nucleobase sequence of miR-21, or a precursor thereof. Incertain such embodiments, a modified oligonucleotide has a nucleobasesequence having no more than two mismatches with respect to thenucleobase sequence of miR-21, or a precursor thereof. In certain suchembodiments, the mismatched nucleobases are contiguous. In certain suchembodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length of miR-21. In certain suchembodiments, the nucleobase of an additional nucleoside is complementaryto a nucleobase of the miR-21 stem-loop sequence. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is one greater than the length of miR-21. In certainsuch embodiments, the additional nucleoside is at the 5′ terminus of anoligonucleotide. In certain such embodiments, the additional nucleosideis at the 3′ terminus of an oligonucleotide. In certain embodiments, thenumber of linked nucleosides of a modified oligonucleotide is twogreater than the length of miR-21. In certain such embodiments, the twoadditional nucleosides are at the 5′ terminus of an oligonucleotide. Incertain such embodiments, the two additional nucleosides are at the 3′terminus of an oligonucleotide. In certain such embodiments, oneadditional nucleoside is located at the 5′ terminus and one additionalnucleoside is located at the 3′ terminus of an oligonucleotide. Incertain embodiments, a region of the oligonucleotide may be fullycomplementary to the nucleobase sequence of miR-21, but the entiremodified oligonucleotide is not fully complementary to miR-21. Forexample, a modified oligonucleotide consisting of 24 linked nucleosides,where the nucleobases of nucleosides 1 through 22 are each complementaryto a corresponding position of miR-21 that is 22 nucleobases in length,has a 22 nucleoside portion that is fully complementary to thenucleobase sequence of miR-21 and approximately 92% overallcomplementarity to the nucleobase sequence of miR-21.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide consists of 7 to 10linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 7 to 12 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 8 to 12 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 8 to 30linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 12 to 30 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 15 to 30 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 12 to 25linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 15 to 25 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 12 to 19 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 15 to 19linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 12 to 16 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 15 to 16 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 19 to 24linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 21 to 24 linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 7 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 8 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 9 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 10 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 11 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 12 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 13 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 14 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 15 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 16 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 17 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 18 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 19 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 20 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 21 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 22 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 23 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 24 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 25 linkednucleosides.

The nucleobase sequences set forth herein, including but not limited tothose found in the examples and in the sequence listing, are independentof any modification to the nucleic acid. As such, nucleic acids definedby a SEQ ID NO may comprise, independently, one or more modifications toone or more sugar moieties, to one or more internucleoside linkages,and/or to one or more nucleobases.

Although the sequence listing accompanying this filing identifies eachnucleobase sequence as either “RNA” or “DNA” as required, in practice,those sequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides issomewhat arbitrary. For example, a modified oligonucleotide comprising anucleoside comprising a 2′-OH sugar moiety and a thymine base could bedescribed as a DNA having a modified sugar (2′-OH for the natural 2′-Hof DNA) or as an RNA having a modified base (thymine (methylated uracil)for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligonucleotide having the nucleobase sequence “ATCGATCG” encompassesany oligonucleotide having such nucleobase sequence, whether modified orunmodified, including, but not limited to, such compounds comprising RNAbases, such as those having sequence “AUCGAUCG” and those having someDNA bases and some RNA bases such as “AUCGATCG” and oligonucleotideshaving other modified bases, such as “AT^(me)CGAUCG,” wherein ^(me)Cindicates a 5-methylcytosine. Similarly, an oligonucleotide having thenucleobase sequence “AUCGAUCG” encompasses any oligonucleotide havingsuch nucleobase sequence, whether modified or unmodified, including, butnot limited to, such compounds comprising DNA bases, such as thosehaving sequence “ATCGATCG” and those having some DNA bases and some RNAbases such as “AUCGATCG” and oligonucleotides having other modifiedbases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a5-methylcytosine.

Certain Synthesis Methods

Modified oligonucleotides may be made with automated, solid phasesynthesis methods known in the art. During solid phase synthesis,phosphoramidite monomers are sequentially coupled to a nucleoside thatis covalently linked to a solid support. This nucleoside is the 3′terminal nucleoside of the modified oligonucleotide. Typically, thecoupling cycle comprises four steps: detritylation (removal of a5′-hydroxyl protecting group with acid), coupling (attachment of anactivated phosphoroamidite to the support bound nucleoside oroligonucleotide), oxidation or sulfurization (conversion of a newlyformed phosphite trimester with an oxidizing or sulfurizing agent), andcapping (acetylation of unreacted 5′-hydroxyl groups). After the finalcoupling cycle, the solid support-bound oligonucleotide is subjected toa detritylation step, followed by a cleavage and deprotection step thatsimultaneously releases the oligonucleotide from the solid support andremoves the protecting groups from the bases. The solid support isremoved by filtration, the filtrate is concentrated and the resultingsolution is tested for identity and purity. The oligonucleotide is thenpurified, for example using a column packed with anion-exchange resin.

GalNAc-conjugated modified oligonucleotides may be made with automatedsolid phase synthesis, similar to the solid phase synthesis thatproduced unconjugated oligonucleotides. During the synthesis ofGalNAc-conjugated oligonucleotides, the phosphoramidite monomers aresequentially coupled to a GalNAc conjugate which is covalently linked toa solid support. The synthesis of GalNAc conjugates and GalNAc conjugatesolid support is described, for example in U.S. Pat. No. 8,106,022,which is herein incorporated by reference in its entirety for thedescription of the synthesis of carbohydrate-containing conjugates,including conjugates comprising one or more GalNAc moieties, and of thesynthesis of conjugate covalently linked to solid support.

Provided herein are processes of making a GalNAc-conjugated modifiedoligonucleotide having the structure shown in formula (IV):

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide; comprising the steps of:

-   providing a solid support comprising a conjugate as shown in formula    IV;

-   deprotecting the DMT group under conditions effective to produce a    reactive hydroxyl;-   performing sequential phosphoramidite coupling steps to form N_(m);-   performing sequential phosphoramidite coupling steps to form MO;-   and releasing the conjugated modified oligonucleotide from the solid    support.

Certain Modifications

In certain embodiments, oligonucleotides provided herein may compriseone or more modifications to a nucleobase, sugar, and/or internucleosidelinkage, and as such is a modified oligonucleotide. A modifiednucleobase, sugar, and/or internucleoside linkage may be selected overan unmodified form because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for other oligonucleotidesor nucleic acid targets and increased stability in the presence ofnucleases.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleosides. In certain such embodiments, a modified nucleosideis a stabilizing nucleoside. An example of a stabilizing nucleoside is asugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modifiednucleoside. In certain such embodiments, the sugar-modified nucleosidescan further comprise a natural or modified heterocyclic base moietyand/or a natural or modified internucleoside linkage and may includefurther modifications independent from the sugar modification. Incertain embodiments, a sugar modified nucleoside is a 2′-modifiednucleoside, wherein the sugar ring is modified at the 2′ carbon fromnatural ribose or 2′-deoxy-ribose.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In certain suchembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In certain embodiments, the bicyclic sugar moiety comprises from1 to 4 linked biradical groups. In certain embodiments, the bicyclicsugar moiety comprises 2 or 3 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. Examples of such 4′ to 2′ sugar substituents, include, but arenot limited to: —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—;4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′; 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-C—H(CH₂OCH₃)—O-2′,and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul.15, 2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂-O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprise 1or from 2 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(X)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising such bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. In certain embodiments, bicyclicnucleosides include, but are not limited to, (A) α-L-Methyleneoxy(4′-CH₂—O-2′) BNA; (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA; (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA; (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA;(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA; (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt);(G) methylene-thio (4′-CH₂—S-2′) BNA; (H) methylene-amino(4′-CH2—N(R)-2′) BNA; (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA;(J) c-MOE (4′-CH₂—OMe-2′) BNA and (K) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from halo, allyl, amino, azido, SH, CN,OCN, CF₃, OCF₃, O—, S—, or N(R_(m))-alkyl; O—, S—, or N(R_(m))-alkenyl;O—, S— or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂-O—N(R_(m))(R_(n))or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with one or more substituent groups independently selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂),thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF_(3,) O—CH₃,O(CH₂)₃NH₂, CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), —O(CH₂)₂0(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N—(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In certain embodiments, a sugar-modified nucleosideis a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-O replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide comprises one or moreinternucleoside modifications. In certain such embodiments, eachinternucleoside linkage of a modified oligonucleotide is a modifiedinternucleoside linkage. In certain embodiments, a modifiedinternucleoside linkage comprises a phosphorus atom.

In certain embodiments, a modified oligonucleotide comprises at leastone phosphorothioate internucleoside linkage. In certain embodiments,each internucleoside linkage of a modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified internucleoside linkage does notcomprise a phosphorus atom. In certain such embodiments, aninternucleoside linkage is formed by a short chain alkyl internucleosidelinkage. In certain such embodiments, an internucleoside linkage isformed by a cycloalkyl internucleoside linkages. In certain suchembodiments, an internucleoside linkage is formed by a mixed heteroatomand alkyl internucleoside linkage. In certain such embodiments, aninternucleoside linkage is formed by a mixed heteroatom and cycloalkylinternucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more short chainheteroatomic internucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more heterocyclicinternucleoside linkages. In certain such embodiments, aninternucleoside linkage has an amide backbone. In certain suchembodiments, an internucleoside linkage has mixed N, O, S and CH₂component parts.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified oligonucleotidecomprises one or more 5-methylcytosines. In certain embodiments, eachcytosine of a modified oligonucleotide comprises a 5-methylcytosine.

In certain embodiments, a modified nucleobase is selected from5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certainembodiments, a modified nucleobase is selected from 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certainembodiments, a modified nucleobase is selected from 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

Certain Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprisingoligonucleotides. In certain embodiments, such pharmaceuticalcompositions are used for the treatment of fibrosis, kidney disease, andcancer. In certain embodiments, a pharmaceutical composition providedherein comprises a compound described herein.

Suitable administration routes include, but are not limited to, oral,rectal, transmucosal, intestinal, enteral, topical, suppository, throughinhalation, intrathecal, intracardiac, intraventricular,intraperitoneal, intranasal, intraocular, intratumoral, and parenteral(e.g., intravenous, intramuscular, intramedullary, and subcutaneous). Incertain embodiments, pharmaceutical intrathecals are administered toachieve local rather than systemic exposures. For example,pharmaceutical compositions may be injected directly in the area ofdesired effect (e.g., into the liver or kidney).

In certain embodiments, a pharmaceutical composition is administered inthe form of a dosage unit (e.g., tablet, capsule, bolus, etc.). In someembodiments, a pharmaceutical compositions comprises a modifiedoligonucleotide at a dose within a range selected from 25 mg to 800 mg,25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mgto 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mgto 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mgto 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. Incertain embodiments, such pharmaceutical compositions comprise amodified oligonucleotide in a dose selected from 25 mg, 30 mg, 35 mg, 40mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580 mg, 585mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625 mg, 630mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg, 675mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg. Incertain such embodiments, a pharmaceutical composition of the comprisesa dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg,100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg,700 mg, and 800 mg.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedmodified oligonucleotide that is reconstituted with a suitable diluent,e.g., sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of a modified oligonucleotide which has been preparedin water for injection, or in saline for injection, adjusted to pH7.0-9.0 with acid or base during preparation, and then lyophilized. Thelyophilized modified oligonucleotide may be 25-800 mg of anoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof modified lyophilized oligonucleotide. Further, in some embodiments,the lyophilized modified oligonucleotide is an amount of anoligonucleotide within a range selected from 25 mg to 800 mg, 25 mg to700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. Thelyophilized drug product may be packaged in a 2 mL Type I, clear glassvial (ammonium sulfate-treated), stoppered with a bromobutyl rubberclosure and sealed with an aluminum FLIP-OFF® overseal.

In certain embodiments, the pharmaceutical compositions provided hereinmay additionally contain other adjunct components conventionally foundin pharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In one method, the nucleic acid is introduced into preformedliposomes or lipoplexes made of mixtures of cationic lipids and neutrallipids. In another method, DNA complexes with mono- or poly-cationiclipids are formed without the presence of a neutral lipid. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to a particular cell or tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to fat tissue. In certain embodiments, a lipidmoiety is selected to increase distribution of a pharmaceutical agent tomuscle tissue.

In certain embodiments, INTRALIPID is used to prepare a pharmaceuticalcomposition comprising an oligonucleotide. Intralipid is fat emulsionprepared for intravenous administration. It is made up of 10% soybeanoil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water forinjection. In addition, sodium hydroxide has been added to adjust the pHso that the final product pH range is 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided hereincomprises a polyamine compound or a lipid moiety complexed with anucleic acid. In certain embodiments, such preparations comprise one ormore compounds each individually having a structure defined by formula(Z) or a pharmaceutically acceptable salt thereof,

wherein each X^(a) and X^(b), for each occurrence, is independently C₁₋₆alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, whereinat least n+2 of the R moieties in at least about 80% of the molecules ofthe compound of formula (Z) in the preparation are not H; m is 1, 2, 3or 4; Y is O, NR², or S; R¹ is alkyl, alkenyl, or alkynyl; each of whichis optionally substituted with one or more substituents; and R² is H,alkyl, alkenyl, or alkynyl; each of which is optionally substituted eachof which is optionally substituted with one or more substituents;provided that, if n=0, then at least n+3 of the R moieties are not H.Such preparations are described in PCT publication WO/2008/042973, whichis herein incorporated by reference in its entirety for the disclosureof lipid preparations. Certain additional preparations are described inAkinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), which isherein incorporated by reference in its entirety for the disclosure oflipid preparations.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein isprepared using known techniques, including, but not limited to mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or tableting processes.

In certain embodiments, a pharmaceutical composition provided herein isa liquid (e.g., a suspension, elixir and/or solution). In certain ofsuch embodiments, a liquid pharmaceutical composition is prepared usingingredients known in the art, including, but not limited to, water,glycols, oils, alcohols, flavoring agents, preservatives, and coloringagents.

In certain embodiments, a pharmaceutical composition provided herein isa solid (e.g., a powder, tablet, and/or capsule). In certain of suchembodiments, a solid pharmaceutical composition comprising one or moreoligonucleotides is prepared using ingredients known in the art,including, but not limited to, starches, sugars, diluents, granulatingagents, lubricants, binders, and disintegrating agents.

In certain embodiments, a pharmaceutical composition provided herein isformulated as a depot preparation. Certain such depot preparations aretypically longer acting than non-depot preparations. In certainembodiments, such preparations are administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided hereincomprises a sustained-release system. A non-limiting example of such asustained-release system is a semi-permeable matrix of solid hydrophobicpolymers. In certain embodiments, sustained-release systems may,depending on their chemical nature, release pharmaceutical agents over aperiod of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain of such embodiments, apharmaceutical composition is formulated by combining one or morecompounds comprising a modified oligonucleotide with one or morepharmaceutically acceptable carriers. Certain of such carriers enablepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. In certain embodiments, pharmaceuticalcompositions for oral use are obtained by mixing oligonucleotide and oneor more solid excipient. Suitable excipients include, but are notlimited to, fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certainembodiments, such a mixture is optionally ground and auxiliaries areoptionally added. In certain embodiments, pharmaceutical compositionsare formed to obtain tablets or dragee cores. In certain embodiments,disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. Incertain such embodiments, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oraladministration are push-fit capsules made of gelatin. Certain of suchpush-fit capsules comprise one or more pharmaceutical agents of thepresent invention in admixture with one or more filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In certain embodiments,pharmaceutical compositions for oral administration are soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In certain soft capsules, one or more pharmaceutical agents ofthe present invention are be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared forbuccal administration. Certain of such pharmaceutical compositions aretablets or lozenges formulated in conventional manner.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition is prepared foradministration by inhalation. Certain of such pharmaceuticalcompositions for inhalation are prepared in the form of an aerosol sprayin a pressurized pack or a nebulizer. Certain of such pharmaceuticalcompositions comprise a propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In certain embodiments using a pressurized aerosol,the dosage unit may be determined with a valve that delivers a meteredamount. In certain embodiments, capsules and cartridges for use in aninhaler or insufflator may be formulated. Certain of such formulationscomprise a powder mixture of a pharmaceutical agent of the invention anda suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition is prepared forrectal administration, such as a suppositories or retention enema.Certain of such pharmaceutical compositions comprise known ingredients,such as cocoa butter and/or other glycerides.

In certain embodiments, a pharmaceutical composition is prepared fortopical administration. Certain of such pharmaceutical compositionscomprise bland moisturizing bases, such as ointments or creams.Exemplary suitable ointment bases include, but are not limited to,petrolatum, petrolatum plus volatile silicones, and lanolin and water inoil emulsions. Exemplary suitable cream bases include, but are notlimited to, cold cream and hydrophilic ointment.

In certain embodiments, a pharmaceutical composition provided hereincomprises a modified oligonucleotide in a therapeutically effectiveamount. In certain embodiments, the therapeutically effective amount issufficient to prevent, alleviate or ameliorate symptoms of a disease orto prolong the survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotides providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Certain Routes of Administration

In certain embodiments, administering to a subject comprises parenteraladministration. In certain embodiments, administering to a subjectcomprises intravenous administration. In certain embodiments,administering to a subject comprises subcutaneous administration.

In certain embodiments, administering to a subject comprisesintraarterial administration. In certain embodiments, administering to asubject comprises intracardial administration. Suitable means forintracardial administration include the use of a catheter, oradministration during open heart surgery. In certain embodiments,administration comprises use of a stent.

In certain embodiments, administration includes pulmonaryadministration. In certain embodiments, pulmonary administrationcomprises delivery of aerosolized oligonucleotide to the lung of asubject by inhalation. Following inhalation by a subject of aerosolizedoligonucleotide, oligonucleotide distributes to cells of both normal andinflamed lung tissue, including alveolar macrophages, eosinophils,epithelium, blood vessel endothelium, and bronchiolar epithelium. Asuitable device for the delivery of a pharmaceutical compositioncomprising a modified oligonucleotide includes, but is not limited to, astandard nebulizer device. Formulations and methods for modulating thesize of droplets using nebulizer devices to target specific portions ofthe respiratory tract and lungs are well known to those skilled in theart. Additional suitable devices include dry powder inhalers or metereddose inhalers.

In certain embodiments, pharmaceutical compositions are administered toachieve local rather than systemic exposures. For example, pulmonaryadministration delivers a pharmaceutical composition to the lung, withminimal systemic exposure.

Additional suitable administration routes include, but are not limitedto, oral, rectal, transmucosal, intestinal, enteral, topical,transdermal, suppository, intrathecal, intraventricular,intraperitoneal, intranasal, intraocular, intramuscular, intramedullary,and intratumoral.

Certain Compounds

Provided herein are compounds comprising a modified oligonucleotidehaving certain nucleoside patterns, and uses of these compounds tomodulate the activity, level or expression of a target nucleic acid. Incertain embodiments, the compound comprises an oligonucleotide. Incertain such embodiments, the compound consists of an oligonucleotide.In certain embodiments, the oligonucleotide is a modifiedoligonucleotide. In certain embodiments, a modified oligonucleotide iscomplementary to a small non-coding RNA. In certain embodiments, thesmall non-coding RNA is miR-21.

In certain such embodiments, the compound comprises a modifiedoligonucleotide hybridized to a complementary strand, i.e. the compoundcomprises a double-stranded oligomeric compound. In certain embodiments,the hybridization of a modified oligonucleotide to a complementarystrand forms at least one blunt end. In certain such embodiments, thehybridization of a modified oligonucleotide to a complementary strandforms a blunt end at each terminus of the double-stranded oligomericcompound. In certain embodiments, a terminus of a modifiedoligonucleotide comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of the complementarystrand. In certain embodiments, the one or more additional nucleosidesare at the 5′ terminus of an oligonucleotide. In certain embodiments,the one or more additional nucleosides are at the 3′ terminus of anoligonucleotide. In certain embodiments, at least one nucleobase of anucleoside of the one or more additional nucleosides is complementary tothe target RNA. In certain embodiments, each nucleobase of each one ormore additional nucleosides is complementary to the target RNA. Incertain embodiments, a terminus of the complementary strand comprisesone or more additional linked nucleosides relative to the number oflinked nucleosides of an oligonucleotide. In certain embodiments, theone or more additional linked nucleosides are at the 3′ terminus of thecomplementary strand. In certain embodiments, the one or more additionallinked nucleosides are at the 5′ terminus of the complementary strand.In certain embodiments, two additional linked nucleosides are linked toa terminus In certain embodiments, one additional nucleoside is linkedto a terminus.

In certain embodiments, the compound comprises a modifiedoligonucleotide conjugated to one or more moieties which enhance theactivity, cellular distribution or cellular uptake of the resultingantisense oligonucleotides. In certain such embodiments, the moiety is acholesterol moiety. In certain embodiments, the moiety is a lipidmoiety. Additional moieties for conjugation include carbohydrates,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. In certainembodiments, a conjugate group is attached directly to anoligonucleotide. In certain embodiments, a conjugate group is attachedto a modified oligonucleotide by a linking moiety selected from amino,hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triplebonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoicacid (AHEX or AHA), substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10alkynyl. In certain such embodiments, a substituent group is selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Suitable cap structures include a 4′,5′-methylene nucleotide, a1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, acarbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, anL-nucleotide, an alpha-nucleotide, a modified base nucleotide, aphosphorodithioate linkage, a threo-pentofuranosyl nucleotide, anacyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotidemoiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotidemoiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridgingmethylphosphonate moiety, and a non-bridging methylphosphonate moiety5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecylphosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotidemoiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety.

Certain Kits

The present invention also provides kits. In some embodiments, the kitscomprise one or more compounds of the invention comprising a modifiedoligonucleotide, wherein the nucleobase sequence of the oligonucleotideis complementary to the nucleobase sequence of miR-21. The compoundscomplementary to miR-21 can have any of the nucleoside patternsdescribed herein. In some embodiments, the compounds complementary tomiR-21 can be present within a vial. A plurality of vials, such as 10,can be present in, for example, dispensing packs. In some embodiments,the vial is manufactured so as to be accessible with a syringe. The kitcan also contain instructions for using the compounds complementary tomiR-21.

In some embodiments, the kits may be used for administration of thecompound complementary to miR-21 to a subject. In such instances, inaddition to compounds complementary to miR-21, the kit can furthercomprise one or more of the following: syringe, alcohol swab, cottonball, and/or gauze pad. In some embodiments, the compounds complementaryto miR-21 can be present in a pre-filled syringe (such as a single-dosesyringes with, for example, a 27 gauge, ½ inch needle with a needleguard), rather than in a vial. A plurality of pre-filled syringes, suchas 10, can be present in, for example, dispensing packs. The kit canalso contain instructions for administering the compounds complementaryto miR-21.

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing modified oligonucleotides of the present invention in anexperimental model. Those having skill in the art are able to select andmodify the protocols for such experimental models to evaluate apharmaceutical agent of the invention.

Generally, modified oligonucleotides are first tested in cultured cells.Suitable cell types include those that are related to the cell type towhich delivery of a modified oligonucleotide is desired in vivo. Forexample, suitable cell types for the study of the methods describedherein include primary or cultured cells.

In certain embodiments, the extent to which a modified oligonucleotideinterferes with the activity of miR-21 is assessed in cultured cells. Incertain embodiments, inhibition of microRNA activity may be assessed bymeasuring the levels of the microRNA. Alternatively, the level of apredicted or validated microRNA-regulated transcript may be measured. Aninhibition of microRNA activity may result in the increase in themiR-21-regulated transcript, and/or the protein encoded bymiR-21-regulated transcript. Further, in certain embodiments, certainphenotypic outcomes may be measured.

Several animal models are available to the skilled artisan for the studyof miR-21 in models of human disease. For example, inhibitors of miR-21may be studied in models of cancer, such as orthotopic xenograft models,toxin-induced cancer models, or genetically-induced cancer models. Insuch cancer models, the studies may be performed to evaluate the effectsof inhibitors of miR-21 on tumor size, tumor number, overall survivaland/or progression-free survival.

The effects of inhibitors of miR-21 on cardiac function and fibrosis maybe studied in models of transaortic banding or myocardial infarction,each of which induces abnormal cardiac function and fibrosis. Models ofkidney fibrosis include unilateral ureteral obstruction andischemia/reperfusion injury. During early time points, the kidneyischemia reperfusion injury model may be used as a model for acutekidney injury, while later time points serve as a model for kidneyfibrosis. An additional model of kidney fibrosis is aristolochicacid-induced fibrosis model. Liver fibrosis models are induced by, forexample, carbon tetrachloride intoxication or bile duct ligation. Liverfibrosis may also be induced by a methionine and choline deficient diet,which results in steatotic liver with associated fibrosis. The effectsof miR-21 on lung fibrosis may be studied, for example, in a model ofbleomycin-induced pulmonary fibrosis or in mice that overexpress TGF-βin the lung. Wound healing models are also available to the skilledartisan, for example the C57B1/KsJ-db/db mice, which exhibit severalcharacteristics of adult onset diabetes, such as markedly delayed woundclosure.

An additional animal model includes a mouse or canine Alport Syndromemodel. An example of a mouse model of Alport Syndrome is the Co14a3knockout mouse.

Certain Quantitation Assays

The effects of antisense inhibition of miR-21 following theadministration of modified oligonucleotides may be assessed by a varietyof methods known in the art. In certain embodiments, these methods arebe used to quantitate microRNA levels in cells or tissues in vitro or invivo. In certain embodiments, changes in microRNA levels are measured bymicroarray analysis. In certain embodiments, changes in microRNA levelsare measured by one of several commercially available PCR assays, suchas the TaqMan® MicroRNA Assay (Applied Biosystems). In certainembodiments, antisense inhibition of miR-21 is assessed by measuring themRNA and/or protein level of a target of miR-21. Antisense inhibition ofmiR-21 generally results in the increase in the level of mRNA and/orprotein of a target of the microRNA.

Target Engagement Assay

Modulation of microRNA activity with an anti-miR or microRNA mimic maybe assessed by measuring target engagement. In certain embodiments,target engagement is measured by microarray profiling of mRNAs. Thesequences of the mRNAs that are modulated (either increased ordecreased) by the anti-miR or microRNA mimic are searched for microRNAseed sequences, to compare modulation of mRNAs that are targets of themicroRNA to modulation of mRNAs that are not targets of the microRNA. Inthis manner, the interaction of the anti-miR with miR-21, or miR-21mimic with its targets, can be evaluated. In the case of an anti-miR,mRNAs whose expression levels are increased are screened for the mRNAsequences that comprise a seed match to the microRNA to which theanti-miR is complementary.

EXAMPLES

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. Those of ordinaryskill in the art will readily adopt the underlying principles of thisdiscovery to design various compounds without departing from the spiritof the current invention.

Example 1 Conjugated Anti-miR-21 Modified Oligonucleotides

Anti-miR-21 modified oligonucleotides were conjugated to aGalNAc-containing moiety, to determine whether the conjugation wouldimprove the potency of the oligonucleotides.

GalNAc-containing compounds were formed by conjugating the structure inFIG. 1 to the 3′ end of the 36731 modified oligonucleotide. In compound40601, the GalNAc-containing moiety is linked to the 3′-terminalnucleoside of 36731 through a β-D-deoxynucleoside, with a phosphodiester(PO) linkage between the 3′-terminal nucleoside of 36731 and theβ-D-deoxynucleoside and a phosphodiester (PO) linkage between theβ-D-deoxynucleoside (β-D-deoxyadenosine (A)) and the GalNAc-containingmoiety, as shown in FIG. 2A, where X₂ is a phosphodiester linkage, m is1, N_(m) is a β-D-deoxynucleoside (A), X₁ is a phosphodiester linkage,and MO is compound 36731. In compound 40379, the GalNAc-containingmoiety is linked to the 3′-terminal nucleoside of 36731 through aphosphodiester (PO) linkage between the 3′-terminal nucleoside of 36731and the GalNAc-containing moiety, as shown in FIG. 2C, where X is aphosphodiester linkage, and MO is compound 36731.

40601: (SEQ ID NO: 56)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S)-PO-A-PO-GalNAc40379: (SEQ ID NO: 56)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S)-PO-GalNAc

Liver concentrations of 36731 and 40601 were measured at 48 hours and168 hours after a single subcutaneous dose of compound in three to fivewild-type C7/B16 mice. The modified oligonucleotide portion of 36731 wasdosed at 1 mg/kg, 3 mg/kg, and 10 mg/kg, and 40601 was dosed at 0.3mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg. Each sample was subjected toliquid chromatography tandem mass spectrometry (LC-MS/MS) to measureoligonucleotide lengths and amounts. As shown in Table A and FIG. 3A,liver concentrations of modified oligonucleotide were significantlyhigher following administration of 40601 than following administrationof a similar dose of 36731, at both time points. Each concentration of40601 shown in FIG. 3A and Table A is the total concentration of allmodified oligonucleotide-containing species detected by LC-MS/MS.Conjugation of modified oligonucleotide resulted in a dose-proportionalincrease in compound in the liver.

Metabolism of 40601 to release modified oligonucleotide 36731 was alsoevaluated. It was found that by 48 hours after a single subcutaneousadministration of compound 40601, the dominant species identified byLC-MS/MS was 36731. Table A and FIG. 3B show the concentration ofmodified oligonucleotide 36731 in mouse livers following a singlesubcutaneous administration of compound 36731 or compound 40601. Table Ashows the percentage of 36731 species detected in mouse liver followingadministration of 40601 (CV %=coefficient of variance).

TABLE A Concentration of 36731 and 40601 in mouse liver after single SCdose Total 40601 Mean compound 36731 Time after Compound Dose detecteddetected dosing dosed (μg/kg) (μg/kg) (μg/kg) CV % % 36731 N  48 hours36731 1 2.9 6 5 3 15.4 18.3 3 10 28.8 14.5 5 40601 0.3 5.5 3.1 5.2 57 51 10.6 6.3 15.4 60 5 3 30.9 17.5 14 57 5 10 57.6 32.7 13.7 57 5 168hours 36731 1 1.9 32.1 4 3 7.6 8.9 5 10 18.6 28 5 40601 0.3 5.5 2.9 4.554 5 1 7.4 5.7 10.2 78 5 3 17.2 13.1 15.7 76 5 10 43.9 33.4 7.8 76 5

Liver concentration of 40379 was also measured at 48 hours and 168 hoursafter a single subcutaneous dose of compound in five wild-type C7/B16mice. The the compound was dosed at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10mg/kg. Each sample was subjected to liquid chromatography tandem massspectrometry (LC-MS/MS) to measure oligonucleotide lengths and amounts.As was observed for compound 40601, the liver concentrations of compoundwere significantly higher following administration of 40379 thanfollowing administration of a similar dose of 36731, at both timepoints. Conjugation of modified oligonucleotide resulted in adose-proportional increase in compound in the liver. Metabolism of 40379to release modified oligonucleotide 36731 was also evaluated. It wasfound that by 48 hours after a single subcutaneous administration ofcompound 40601, compound 36371 was present, but at lower concentrationsrelative to the amount of compound 36371 present followingadministration of compound 40379 (data not shown). Whereas at least 50%of compound 40601 was present as compound 36731 after 48 or 168 hours,approximately 15 to 30% of compound 40379 was present as compound 36731.Thus, while compound 40379 does undergo some metabolism that results inthe release of unconjugated compound 36731, the release of unconjugatedcompound is less than that observed for compound 40601. These datasuggest that the presence of the PO-A-PO linker facilitates release ofthe unconjugated modified oligonucleotide from the GalNAc-containingcompound.

Example 2 Pharmacodynamic Activity of Conjugated Anti-miR-21 ModifiedOligonucleotides

To assess the effect of inhibition of miR-21 on known mRNA targets,de-repression of SPG20, Rnf167 and Taf7 in normal mouse liver wasmeasured following a single dose of 36731 (1 and 10 mg/kg) or a singledose of 40601 (0.1, 1 and 10 mg/kg) administered to wild-type mice.Livers were harvested 4 or 7 days after administration. As shown inTable B, modest target gene derepression was observed for SPG20 and Taf7seven days after a single dose of 36731, while Rnf167 was derepressed atboth time points at the highest dose. Single dose administration of40601 showed improved target derepression of both SPG20 and Taf7 at bothtime points and similar derepression of Rnf167. Improved targetderepression included both larger fold change in target gene expressionand earlier onset of derepression.

TABLE B Derepression of miR-21 target genes in normal liver followingadministration of 36731 or 40601 Time Average target gene levelTreatment Point Dose SPG20 Rnf167 Taf7 Vehicle 1.00 1.00 1.00  1 mg/kg0.98 1.07 0.96 Day 4 10 mg/kg 0.97 1.29 1.02 36731  1 mg/kg 1.11 0.911.21 Day 7 10 mg/kg 1.25 1.11 1.16 Day 2 10 mg/kg 1.36 1.24 1.28 0.1mg/kg  1.16 0.97 1.19 Day 4  1 mg/kg 1.32 0.97 1.12 40601 10 mg/kg 1.441.27 1.60 0.1 mg/kg  1.23 0.65 1.75 Day 7  1 mg/kg 1.70 1.04 2.49 10mg/kg 1.53 1.32 3.28

Example 3 In Vivo Efficacy of Conjugated Anti-miR-21 ModifiedOligonucleotides

A liver-specific doxycycline-regulated oncogene expression system wasused to model hepatocellular carcinoma (HCC) in the mouse. In thismodel, transgenic mice express the oncogene H-rasG12V under the controlof a doxycycline-repressable, liver-specific promoter (Tet-o-H-rasG12V;LAP-TTA; See, for example, Lim et al., Hepatology, 2013). Whendoxycycline is removed, H-rasG12V transgene expression is activated inthe liver and the mice develop liver tumors. It was confirmed thatexpression of the GalNAc receptors ASGR1 and ASGR2 remains high for atleast 6 weeks following removal of doxycycline, while miR-21 expressionincreases with the onset of morphologically detectable disease (data notshown).

In order to demonstrate delivery of compounds 40601 and 36731 to livertumors, the compounds were administered to mice 4 weeks after removal ofdoxycycline, by which time the mice have a significant tumor burden. Theconcentrations of 36731 and 40601 in liver tumor tissue (whole livercontaining tumor) were measured at 168 hours after two doses of compoundin five mice per group. The first dose was given at 0 hours, the seconddose at 72 hours and tumor tissue harvested at 168 hours. Compound 36731was administered at 10 mg/kg, while compound 40601 was administered at0.1, 1, and 10 mg/kg. As shown in Table C, total drug level achieved was80% greater for 40601 as compared to 36731. Release of 36731 modifiedoligonucleotide from compound 40601 was between 44 and 70% of the total.

TABLE C Quantification of 36731 and 40601 in mouse liver tumor tissuefollowing administration Cmpd admin.: 36731 40601 Cmpd detected: 3673136731 All Mean Mean Mean Dose Total dose detected detected % detected(μg/kg) (μg/kg) (μg/kg) CV % (μg/kg) CV % 36731 (μg/kg) CV % 0.1 BIW 0.21.7 18 63 2.7 28   1 BIW 2 4.9 35 70 7 32  10 BIW 20 23.3 38 17.9 35 4440.5 33

Target derepression for the three miR-21 target genes evaluated innormal liver was also evaluated in the liver tumor tissue. Althoughstatistical significance was not achieved, there was a trend towardsderepression in treated liver tumor tissue as compared to vehicle. TableD shows derepression of SPG21, Rnf167, and Taf7 in liver tumor tissuefollowing administration of compound 40601 or compound 36731.

TABLE D Derepression of miR-21 target genes in liver tumor tissuefollowing administration of 36731 or 40601 Average target gene levelTreatment SPG20 Rnf167 Taf7 Vehicle 1.00 1.00 1.00 36731 10 mg/kg 1.011.37 1.72 40601 10 mg/kg 1.24 1.52 1.56 40601 1 mg/kg 1.31 1.50 1.1240601 0.1 mg/kg 0.84 1.28 0.78

In addition, AFP levels in liver tumor tissue were evaluated followingadministration of compound 40601 or compound 36731. There was a trendtoward reduced AFP levels in the mice that received a biweekly dose of10 mg/kg 40601.

Next, efficacy of compounds 40601 and 36731 was tested in theTet-o-H-rasG12V;LAP-TTA transgenic mice. Tumor progression was initiatedby removing doxycycline from male mice at 6 weeks of age. After twoweeks off doxycycline, mice were divided into 5 treatment groups:vehicle, 36731 25 mg/kg biweekly (BIW), 40601 25 mg/kg BIW, 40601 25mg/kg once weekly (Q7D), and 40601 5 mg/kg BIW. Mice were treated for 4weeks. Liver morphology (mottled appearance) was used as an indicator oftumor formation and was scored at the end of study by an investigatorblinded to the treatment group. As shown in Table E, the majority (6/8)of the animals in the vehicle group had mottled livers, whileapproximately half (5/9) of the animals in the 25 mg/kg 36731 BIW grouphad mottled livers. A greater reduction in the incidence of mottledappearance was seen in the BIW dosing groups of compound 40601, with 3/8in the 25 mg/kg 40601 BIW group and 2/8 in the 5 mg/kg 40601 BIW groupsshowing mottled livers. Once weekly dosing of 25 mg/kg 40601 resulted ina similar mottled appearance frequency (8/10) to vehicle.

TABLE E Incidence of mottled livers in transgenic mice followingadministration of 36731 or 40601 Total # mottled # not mottled CompoundDose mice livers livers vehicle 8 6 2 36731 25 mg/kg BIW 9 5 4 40601  5mg/kg BIW 8 2 6 40601 25 mg/kg BIW 8 3 5 40601 25 mg/kg Q7D 10 8 2

At the end of the study, liver tumor tissue AFP was assessed as a markerof liver tumors in the samples by Western blot analysis, normalized toβ-actin. As shown in Table F, AFP was significantly reduced by treatmentwith 25 mg/kg 40601 BIW.

TABLE F AFP levels in liver tumors of transgenic mice followingadministration of 36731 or 40601 Treatment Dose Frequency AFP AverageVehicle 0.09 36731 25 mg/kg BIW 0.08 40601 25 mg/kg BIW 0.03 40601  5mg/kg BIW 0.06 40601 25 mg/kg Q7D 0.07Target derepression following 36731 or 40601 administration was assessedin the liver tumor tissue of the transgenic mice. As shown in Table G,both SPG20 and Rnf167 transcripts were derepressed in the end of studysamples from the 36731 and 40601 BIW dosing groups. Taf7 was alsoevaluated but did not show consistent derepression (data not shown).

TABLE G SPG20 and Rnf167 target derepression in liver tumors oftransgenic mice following administration of 36731 or 40601 TreatmentDose Frequency SPG20 Average Rnf167 Average Vehicle 1.00 1.00 36731 25mg/kg BIW 1.75 2.01 40601 25 mg/kg BIW 1.59 2.44 40601  5 mg/kg BIW 1.512.09 40601 25 mg/kg Q7D 1.33 1.78

Finally, tumor drug concentrations were evaluated at the end of thestudy. As shown in Table H, comparable levels of 36731 and total 40601were achieved. Release of 36731 modified oligonucleotide from 40601 wasapproximately 50% of the total at all three doses.

TABLE H Quantification of 36731 and 40601 in liver tumor tissuefollowing administration Cmpd admin.: 36731 40601 Cmpd detect.: 3673136731 All Mean Mean Mean Dose Total dose detected detected % detected(μg/kg) (μg/kg) (μg/kg) CV % (μg/kg) CV % 36731 (μg/kg) CV %  5 BIW ×440 28.4 28 49 58.3 29.4 25 QW ×4 100 37.9 23 41 92.2 24.6 25 BIW ×4 200233.5 37 96.0 31 49 197.1 30.5

To evaluate whether there was impact on efficacy parameters startingtreatment with more advanced disease, a study with a comparable designwas initiated, except treatment was started at 4 weeks after removal ofdoxycycline and the mice were only treated for three weeks. In thisexperiment, the once per week dosing group was not included. AFP andtarget gene assessment was measured at the end of the study. A trendtowards reduced AFP levels in the high dose group of 40601, 25 mg/kgBIW, was observed (data not shown). Target engagement was also observedin the treatment groups, reaching statistical significance with Taf7 at25 mg/kg of 36731 and 40601 (data not shown).

Example 4 Improved Survival of Transgenic Mice Administered ConjugatedAnti-miR-21 Modified Oligonucleotides

To evaluate improvement in survival, Tet-o-H-rasG12V;LAP-TTA transgenicmice are administered vehicle, 25 mg/kg compound 36731, or 25 mg/kgcompound 40601 biweekly, beginning at two weeks after removal ofdoxycycline. The mice receive biweekly administrations of compound untilthe end of the study. On average, mice that receive biweeklyadministrations of 36731 or 40601 are expected to have longer survivaltimes than mice that are administered vehicle. Further, mice thatreceive 40601 are expected to have longer survival times than mice thatreceive 36731.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, GENBANK®accession numbers, and the like) cited in the present application isspecifically incorporated herein by reference in its entirety.

1. A compound having the structure:L_(n)-linker-X—N_(m)—X-MO wherein each L is, independently, a ligand andn is from 1 to 10; each N is, independently, a modified or unmodifiednucleoside and m is from 1 to 5; each X is, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide, wherein a. the modified oligonucleotidecomprises at least 8 contiguous nucleosides of the following nucleosidepattern I in the 5′ to 3′ orientation:(R)_(X)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Q)—N^(Z)wherein each R is, independently, a non-bicyclic nucleoside; X is from 1to 4; each N^(B) is, independently, a bicyclic nucleoside; each N^(Q)is, independently, a non-bicyclic nucleoside; and each N^(Z) is,independently, a modified nucleoside; b. the modified oligonucleotideconsists of 8 to 19 linked nucleosides, and wherein the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern II in the 5′ to 3′ orientation:N^(M)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Q)—N^(Z)wherein N^(M) is, independently, a modified nucleoside that is not abicyclic nucleoside; each N^(B) is, independently, a bicyclicnucleoside; each N^(Q) is, independently, a non-bicyclic nucleoside; andN^(Z) is, independently, a modified nucleoside; c. the modifiedoligonucleotide comprises at least 8 contiguous nucleosides of thefollowing nucleoside pattern III in the 5′ to 3′ orientation:(R)_(X)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Y)—N^(Z)wherein each R is a non-bicyclic nucleoside; X is from 1 to 4; eachN^(B) is a bicyclic nucleoside; each N^(Q) is a non-bicyclic nucleoside;N^(Y) is a modified nucleoside or an unmodified nucleoside; and eachN^(Z) is a modified nucleoside; d. the modified oligonucleotide consistsof 8 to 19 linked nucleosides, and wherein the modified oligonucleotidecomprises at least 8 contiguous nucleosides of the following nucleosidepattern IV in the 5′ to 3′ orientation:N^(M)—N^(B)—N^(Q)—N^(Q)—N^(B)—(N^(Q)—N^(Q)—N^(Q)—N^(B))₃—N^(Y)—N^(Z)wherein N^(M) is a modified nucleoside that is not a bicyclicnucleoside; each N^(B) is a bicyclic nucleoside; each N^(Q) is anon-bicyclic nucleoside; N^(Y) is a modified nucleoside or an unmodifiednucleoside; and N^(Z) is a modified nucleoside; e. the modifiedoligonucleotide consists of 8 to 19 linked nucleosides, and wherein themodified oligonucleotide comprises at least 8 contiguous nucleosides ofthe following nucleoside pattern V in the 5′ to 3′ orientation:N^(M)—N^(B)—(N^(Q)—N^(Q)—N^(B)—N^(B))₄—N^(Z) wherein N^(M) is a modifiednucleoside that is not a bicyclic nucleoside; each N^(B) is a bicyclicnucleoside; each N^(Q) is a non-bicyclic nucleoside; and N^(Z) is amodified nucleoside; f. the modified oligonucleotide consists of 8 to 15linked nucleosides, and wherein the modified oligonucleotide comprisesat least 8 contiguous nucleosides of the following nucleoside pattern VIin the 5′ to 3′ orientation:N^(Q)—N^(B)—N^(B)—N^(Q)—(N^(B)—N^(B)—N^(Q)—N^(Q))₂—N^(B)—N^(Q)—N^(B)wherein each N^(Q) is a non-bicyclic nucleoside; and each N^(B) is abicyclic nucleoside; and/or g. the modified oligonucleotide consists of8 to 19 linked nucleosides, and wherein the modified oligonucleotidecomprises at least 8 contiguous nucleosides of the following nucleosidepattern VII in the 5′ to 3′ orientation:N^(M)—(N^(B)—N^(M)—N^(M))₂—N^(M)—(N^(B)—N^(Q)—N^(Q)—N^(Q))₂—N^(B)—N^(B)—N^(Z)wherein each N^(M) is a modified nucleoside that is not a bicyclicnucleoside; each N^(B) is a bicyclic nucleoside; each N^(Q) is anon-bicyclic nucleoside; and N^(Z) is a modified nucleoside. 2.(canceled)
 3. (canceled)
 4. The compound of claim 1, wherein if n isgreater than 1, L_(n)-linker has the structure:

wherein each L is, independently, a ligand; n is from 1 to 10; S is ascaffold; and Q′ and Q″ are, independently, linking groups. 5.(canceled)
 6. The compound of claim 4, wherein the scaffold links 2, 3,4, or 5 ligands to a modified oligonucleotide.
 7. (canceled)
 8. Thecompound of claim 1, comprising the structure:

wherein: B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,—Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—; MO is the modifiedoligonucleotide; Z, Z′, and Z″ are each independently selected from Oand S; each N is, independently, a modified or unmodified nucleoside; mis from 1 to 5; X is selected from a phosphodiester linkage and aphosphorothioate linkage; Y is a phosphodiester linkage; and the wavyline indicates the connection to the rest of the linker and ligand(s).9. The compound of claim 1, wherein n is from 1 to 5, 1 to 4, 1 to 3, or1 to
 2. 10. (canceled)
 11. The compound of claim 1, wherein at least oneligand is selected from a carbohydrate, cholesterol, a lipid, aphospholipid, an antibody, a lipoprotein, a hormone, a peptide, avitamin, a steroid, and a cationic lipid.
 12. (canceled)
 13. Thecompound of claim 1, wherein at least one ligand is selected fromN-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, andN-iso-butanoyl-galactosamine.
 14. (canceled)
 15. The compound of claim1, wherein the compound has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is themodified oligonucleotide.
 16. The compound of claim 0, wherein at leastone of X₁ and X₂ is a phosphodiester linkage.
 17. The compound of claim0, wherein each of X₁ and X₂ is a phosphodiester linkage.
 18. Thecompound of claim 1, wherein m is 1, 2, 3, 4 or
 5. 19-31. (canceled) 32.The compound of claim 1, wherein the sugar moiety of each N isindependently selected from a β-D-ribose, a β-D-deoxyribose, a2′-O-methoxy sugar, a 2′-O-methyl sugar, a 2′-fluoro sugar, and abicyclic sugar moiety. 33-44. (canceled)
 45. The compound of claim 1,wherein each bicyclic nucleoside is independently selected from an LNAnucleoside, a cEt nucleoside, and an ENA nucleoside.
 46. (canceled) 47.(canceled)
 48. The compound of claim 1, wherein each non-bicyclicnucleoside is independently selected from a β-D-deoxyribonucleoside, a2′-O-methyl, and a 2′-O-methoxyethyl nucleoside. 49-51. (canceled) 52.The compound of claim 1 wherein: i. the compound is a compound of claim1(a), wherein: a. R consists of four linked nucleosidesN^(R1)—N^(R2)—N^(R3)—N^(R4) wherein N^(R1) is a 2′-O-methoxyethylnucleoside and each of N^(R2)—N^(R3)—N^(R4) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside; b. each R is a 2′-O-methoxyethyl nucleoside; X is 1; eachN^(B) is an S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside;and N^(Z) is a 2′-O-methoxyethyl nucleoside; c. each R is a2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEt nucleoside;each N^(Q) is a 2′-O-methoxyethyl nucleoside; and N^(Z) is a2′-O-methoxyethyl nucleoside; d. each R is a 2′-O-methoxyethylnucleoside; X is 1; each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside; e. each R isa 2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an LNA nucleoside;each N^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a2′-O-methoxyethyl nucleoside; or f. each R is a 2′-O-methoxyethylnucleoside; X is 1; each N^(B) is an LNA nucleoside; each N^(Q) is a3-D-deoxyribonucleoside; and N^(Z) is an LNA nucleoside; ii. thecompound is a compound of claim 1(b), wherein: a. N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethylnucleoside; b. N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a 2′-O-methoxyethyl nucleoside; andN^(Z) is a 2′-O-methoxyethyl nucleoside; c. N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; each N is a β-D-deoxyribonucleoside; and N^(Z)is an S-cEt nucleoside; d. N^(M) is a 2′-O-methoxyethyl nucleoside; eachN^(B) is an LNA nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; andN^(Z) is a 2′-O-methoxyethyl nucleoside; or e. N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an LNA nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and N^(Z) is an LNA nucleoside; iii.the compound is a compound of claim 1(c), wherein: a. each R is a2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEt nucleoside;each N^(Q) is aβ-D-deoxyribonucleoside; N^(Y) is aβ-D-deoxyribonucleoside; and N^(Z) is a 2′-O-methoxyethyl nucleoside; b.each R is a 2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEtnucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; N^(Y) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside; or c. each Ris a 2′-O-methoxyethyl nucleoside; X is 1; each N^(B) is an S-cEtnucleoside; each N^(Q) is a β-D-deoxyribonucleoside; N^(Y) is an S-cEtnucleoside; and N^(Z) is an S-cEt nucleoside; d. each R is a2′-O-methoxyethyl nucleoside; X is 1; each NB is an S-cEt nucleoside;each N^(Q) is independently selected from a β-D-deoxyribonucleoside anda 2′-O-methoxyethyl nucleoside; NY is selected from an S-cEt nucleosideand a β-D-deoxyribonucleoside; and NZ is an S-cEt nucleoside; iv. thecompound is a compound of claim 1(d), wherein: a. N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; N^(Y) is a β-D-deoxyribonucleoside;N^(Z) is a 2′-O-methoxyethyl nucleoside; and b. N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; N^(Y) is a β-D-deoxyribonucleoside;and N^(Z) is an S-cEt nucleoside; c. N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) isaβ-D-deoxyribonucleoside; N^(Y) is an S-cEt nucleoside; and N^(Z) is anS-cEt nucleoside; d. N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B)is an S-cEt nucleoside; each N^(Q) is independently selected from aβ-D-deoxyribonucleoside and a 2′-O-methoxyethyl nucleoside; N^(Y) isselected from an S-cEt nucleoside and a β-D-deoxyribonucleoside; andN^(Z) is an S-cEt nucleoside; v. the compound is a compound of claim1(e), wherein: a. N^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) isan S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and N^(Z)is a 2′-O-methoxyethyl nucleoside; vi. the compound is a compound ofclaim 1(f), wherein: a. each N^(B) is an S-cEt nucleoside; and eachN^(Q) is a 2′-O-methoxyethyl nucleoside; b. each N^(B) is an S-cEtnucleoside; and each N^(Q) is a β-D-deoxyribonucleoside; vii. thecompound is a compound of claim 1(g), wherein: a. each N^(M) is a2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is independently selected from a 2′-O-methyl nucleoside and aβ-D-deoxyribonucleoside; and N^(Z) is selected from an S-cEt nucleosideand a 2′-O-methoxyethyl nucleoside; b. each N^(M) is a 2′-O-methoxyethylnucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside; c. each N^(M)is a 2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEt nucleoside;each N^(Q) is independently selected from a 2′-O-methyl nucleoside and aβ-D-deoxyribonucleoside; and N^(Z) is an S-cEt nucleoside; or d. eachN^(M) is a 2′-O-methoxyethyl nucleoside; each N^(B) is an S-cEtnucleoside; each N^(Q) is independently selected from a 2′-O-methylnucleoside and a β-D-deoxyribonucleoside; and N^(Z) is a2′-O-methoxyethyl nucleoside. 53-57. (canceled)
 58. The compound ofclaim 1 having the structure: (SEQ ID NO: 3)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)TA_(S);(SEQ ID NO: 3)A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S);(SEQ ID NO: 8)A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)U_(S)A_(E); or(SEQ ID NO: 7)^(Me)C_(E)A_(S)A_(S)T_(E)C_(S)U_(S)A_(E)A_(E)U_(S)A_(S)A_(E)G_(E)C_(S)T_(E)A_(S);

wherein nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; nucleosides followed by a subscript “S” are S-cEtnucleosides; and ^(Me)C is 5-methyl cytosine. 59-65. (canceled)
 66. Thecompound of claim 1, wherein the modified oligonucleotide consists of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 linkednucleosides of nucleoside pattern I, II, III, IV, V, VI, or VII. 67.(canceled)
 68. (canceled)
 69. The compound of claim 1, wherein thenucleobase sequence of the modified oligonucleotide is selected from SEQID NOs: 3 to 10, wherein each T is independently selected from T and U.70. The compound of claim 1, wherein the modified oligonucleotide has 0,1, 2, or 3 mismatches with respect to the nucleobase sequence of miR-21.71-74. (canceled)
 75. The compound of claim 1, wherein the modifiedoligonucleotide has a structure selected from the structures in Table 1.76. A method of inhibiting the activity of miR-21 comprising contactinga cell with a compound of claim
 1. 77-79. (canceled)
 80. A method oftreating, preventing or delaying the onset of a disease associated withmiR-21 comprising administering to a subject having a disease associatedwith miR-21 a compound of claim
 1. 81. The method of claim 80, whereinthe disease is fibrosis. 82-88. (canceled)
 89. The method of claim 80,wherein the disease is cancer. 90-106. (canceled)